Tgf-beta receptor type ii fusion proteins and uses thereof

ABSTRACT

In certain aspects, the present disclosure relates to TβRII fusion polypeptides comprising a heterologous portion and a truncated, ligand-binding portion of the extracellular domain of TβRII polypeptide useful to selectively antagonize a TβRII ligand. The disclosure further provides compositions and methods for use in treating or preventing TGFβ associated disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/969,957, filed May 3, 2018 (now allowed), which claims the benefit ofpriority from U.S. Provisional Application No. 62/501,229 (now expired),filed on May 4, 2017; from U.S. Provisional Application No. 62/510,422(now expired), filed on May 24, 2017; and from U.S. ProvisionalApplication No. 62/578,674 (now expired), filed on Oct. 30, 2017. Theforegoing applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 29, 2021, isnamed 1848179-0002-121-102 SL.txt and is 113,369 bytes in size.

BACKGROUND OF THE INVENTION

Members of the transforming growth factor-beta (TGFβ) superfamily arepleiotropic cytokines involved in essential cellular functions such asproliferation, differentiation, apoptosis, motility, extracellularmatrix production, tissue remodeling, angiogenesis, immune response,cell adhesion, and also play a key role in pathophysiology of diseasestates as different as chronic inflammatory conditions and cancer.Members of the TGFβ superfamily have been classified into major familygroupings, which include TGFβs, bone morphogenetic proteins (BMP),osteogenic proteins (OP), growth and differentiation factors (GDF),inhibins/activins, mullerian inhibitory substances (MIS) and glialderived neurotrophic factors (GDNF).

TGFβ superfamily members transduce their signals across the plasmamembrane by inducing the formation of heteromeric complexes of specifictype I and type II serine/threonine kinase receptors, which in turnactivate a particular subset of SMAD proteins (some inhibitory and someexcitatory). The SMAD molecule compounds relay the signals into thenucleus where they direct transcriptional responses in concert withother proteins.

Dysfunctional TGFβ superfamily signaling has been linked to severalclinical disorders including cancer, fibrosis, bone diseases, diabeticnephropathy, as well as chronic vascular diseases such asatherosclerosis.

Thus, it is an object of the present disclosure to provide compositionsand methods for modulating TGFβ superfamily signaling.

SUMMARY OF THE INVENTION

In part, the disclosure provides TβRII polypeptide fusion proteins andthe use of such fusion proteins as selective antagonists for TGFβ1 orTGFβ3. As described herein, polypeptides comprising part or all of theTβRII extracellular domain (ECD), with or without additional mutations,bind to and/or inhibit TGFβ1 or TGFβ3 with varying affinities. Inparticular, TβRII polypeptides comprising a heterologous portion (e.g.,an Fc immunoglobulin domain) and a linker of at least 10 amino acids inlength (e.g., a linker having the amino acid sequence of SEQ ID NO: 6)are associated with surprisingly superior TGFβ1 and TGFβ3 bindingproperties as compared to TβRII polypeptides having a shorter linker.Thus, in certain aspects, the disclosure provides TβRII polypeptides foruse in selectively inhibiting TGFβ superfamily associated disorders.

In some embodiments, the disclosure provides for a Transforming GrowthFactor-β Receptor II (TβRII) fusion polypeptide comprising: a) anextracellular domain of a TβRII portion; b) a heterologous portion, andc) a linker portion; wherein the linker is at least 10 amino acids inlength; and wherein the TβRII extracellular domain portion comprises anamino acid sequence at least 80% identical to: i) a sequence beginningat any of positions 23 to 35 of SEQ ID NO: 1 and ending at any ofpositions 153 to 159 of SEQ ID NO: 1 or ii) a sequence beginning at anyof positions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178to 184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellulardomain portion comprises an amino acid sequence at least 80% identicalto a sequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 90% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 95% identical to asequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 97% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence beginning at any of positions23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to 159 ofSEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 80% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 90% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 95% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion comprises an aminoacid sequence at least 97% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence beginning at any of positions23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to 184 ofSEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion comprises an amino acid sequence at least 80% identical to SEQID NO: 18. In some embodiments, the TβRII extracellular domain portioncomprises an amino acid sequence at least 90% identical to SEQ ID NO:18. In some embodiments, the TβRII extracellular domain portioncomprises an amino acid sequence at least 95% identical to SEQ ID NO:18. In some embodiments, the TβRII extracellular domain portioncomprises an amino acid sequence at least 97% identical to SEQ ID NO:18. In some embodiments, the TβRII extracellular domain portioncomprises the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the TβRII extracellular domain portion consists ofan amino acid sequence at least 80% identical to a sequence beginning atany of positions 23 to 35 of SEQ ID NO: 1 and ending at any of positions153 to 159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellulardomain portion consists of an amino acid sequence at least 90% identicalto a sequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 95% identical to a sequence beginning at any ofpositions 23 to 35 of SEQ ID NO: 1 and ending at any of positions 153 to159 of SEQ ID NO: 1. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 97% identical to asequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence beginning at any of positions 23 to 35 of SEQ ID NO: 1 andending at any of positions 153 to 159 of SEQ ID NO: 1. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 80% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 90% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 95% identical to a sequence beginning at any ofpositions 23 to 60 of SEQ ID NO: 2 and ending at any of positions 178 to184 of SEQ ID NO: 2. In some embodiments, the TβRII extracellular domainportion consists of an amino acid sequence at least 97% identical to asequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence beginning at any of positions 23 to 60 of SEQ ID NO: 2 andending at any of positions 178 to 184 of SEQ ID NO: 2. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 80% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 90% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 95% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of an aminoacid sequence at least 97% identical to SEQ ID NO: 18. In someembodiments, the TβRII extracellular domain portion consists of theamino acid sequence of SEQ ID NO: 18. In some embodiments, thepolypeptide comprises an N-terminal leader sequence. In someembodiments, the N-terminal leader sequence comprises the amino acidsequence of any one of SEQ ID NOs: 22-24. In some embodiments, theN-terminal leader sequence comprises the amino acid sequence of SEQ IDNO: 23. In some embodiments, the heterologous portion is animmunoglobulin Fc domain. In some embodiments, the immunoglobulin Fcdomain is a human immunoglobulin Fc domain. In some embodiments, theheterologous portion comprises an amino acid sequence that is at least80% identical to SEQ ID NO: 20. In some embodiments, the heterologousportion comprises an amino acid sequence that is at least 90% identicalto SEQ ID NO: 20. In some embodiments, the linker is less than 25 aminoacids in length. In some embodiments, the linker is between 10 and 25amino acids in length. In some embodiments, the linker is between 15 and25 amino acids in length. In some embodiments, the linker is between 17and 22 amino acids in length. In some embodiments, the linker is 21amino acids in length. In some embodiments, the heterologous portioncomprises an amino acid sequence that is at least 95% identical to SEQID NO: 20. In some embodiments, the heterologous portion comprises anamino acid sequence that is at least 97% identical to SEQ ID NO: 20. Insome embodiments, the heterologous portion comprises the amino acidsequence of SEQ ID NO: 20. In some embodiments, the linker comprises(GGGGS)n, wherein n=>2 (SEQ ID NO: 57). In some embodiments, the linkercomprises (GGGGS)n, wherein n=>3 (SEQ ID NO: 58). In some embodiments,the linker comprises (GGGGS)n, wherein n=>4 (SEQ ID NO: 59). In someembodiments, the linker comprises (GGGGS)_(n), wherein n≠>5 (SEQ ID NO:60). In some embodiments, the linker comprises the amino acid sequenceof SEQ ID NO: 21. In some embodiments, the linker comprises the aminoacid sequence of any one of SEQ ID NOs: 4-7. In some embodiments, thelinker comprises the amino acid sequence of SEQ ID NO: 6. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 80% identical to the amino acid sequence of SEQ ID NO: 11. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 11. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 11. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 11. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 13. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 13. In some embodiments, the polypeptide comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 13. In some embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 13. In some embodiments,the polypeptide comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 50. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 50. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 50. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 50. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 51. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 51. In some embodiments, the polypeptide comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 51. In some embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 51. In some embodiments,the polypeptide comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 53. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 53. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 53. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 53. In some embodiments, the polypeptide comprises an amino acidsequence that is at least 80% identical to the amino acid sequence ofSEQ ID NO: 56. In some embodiments, the polypeptide comprises an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 56. In some embodiments, the polypeptide comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 56. In some embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 56. In some embodiments,the polypeptide comprises an amino acid sequence that is at least 80%identical to the amino acid sequence of SEQ ID NO: 15. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 15. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 15. In someembodiments, the polypeptide comprises the amino acid sequence of SEQ IDNO: 15. In some embodiments, the TβRII polypeptide does not includeamino acids 185-592 of SEQ ID NO: 2. In some embodiments, the TβRIIpolypeptide does not include amino acids 1-22 of SEQ ID NO: 2. In someembodiments, the polypeptide consists of or consists essentially of: a)a TβRII polypeptide portion comprising an amino acid sequence that is atleast 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence ofSEQ ID NO: 18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1additional amino acids; b) a linker portion comprising an amino acidsequence that is at least 85%, 90%, 95%, 97%, or 99% identical to theamino acid sequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1additional amino acids; c) a heterologous portion comprising an aminoacid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical tothe amino acid sequence of SEQ ID NO: 20 and no more than 25, 20, 15,10, 5, 4, 3, 2, or 1 additional amino acids; and d) optionally a leadersequence (e.g., SEQ ID NO: 23). In some embodiments, the polypeptideconsists of or consists essentially of: a) a TβRII polypeptide portioncomprising the amino acid sequence of SEQ ID NO: 18 and no more than 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids; b) a linker portioncomprising the amino acid sequence of SEQ ID NO: 6 and no more than 5,4, 3, 2 or 1 additional amino acids; c) a heterologous portioncomprising the amino acid sequence of SEQ ID NO: 20 and no more than 25,20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids; and d) optionally aleader sequence (e.g., SEQ ID NO: 23). In some embodiments, thepolypeptide comprises: a) an extracellular domain of a TβRII portion;wherein the extracellular domain comprises an amino acid sequence thatis at least 85%, 90%, 95%, 97%, or 99% identical to the sequence of SEQID NO: 18; b) a heterologous portion, wherein the heterologous portioncomprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or99% identical to the sequence of SEQ ID NO: 20; and c) a linker portionconnecting the extracellular domain and the heterologous portion;wherein the linker comprises an amino acid sequence that is at least85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of SEQID NO: 6. In some embodiments, the polypeptide comprises: a) anextracellular domain of a TβRII portion; wherein the extracellulardomain comprises the amino acid sequence of SEQ ID NO: 18; b) aheterologous portion, wherein the heterologous portion comprises theamino acid sequence of SEQ ID NO: 20; and c) a linker portion connectingthe extracellular domain and the heterologous portion; wherein thelinker comprises the amino acid sequence of SEQ ID NO: 6. In someembodiments, the polypeptide comprises an amino acid sequence that is atleast 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence ofSEQ ID NO: 48. In some embodiments, the polypeptide comprises the aminoacid sequence of SEQ ID NO: 48. In some embodiments, the polypeptidedoes not include a leader sequence, or wherein the leader sequence hasbeen removed. In some embodiments, the polypeptide includes one or moremodified amino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.In some embodiments, the polypeptide is glycosylated. In someembodiments, the polypeptide has a glycosylation pattern characteristicof expression of the polypeptide in CHO cells. In some embodiments, thepolypeptide binds human TGFβ1 with an equilibrium dissociation constant(KD) less than 100 pM. In some embodiments, the polypeptide binds humanTGFβ1 with an equilibrium dissociation constant (KD) less than 75 pM. Insome embodiments, the polypeptide binds human TGFβ3 with an equilibriumdissociation constant (KD) less than 60 pM. In some embodiments, thepolypeptide binds human TGFβ3 with an equilibrium dissociation constant(KD) less than 50 pM. In some embodiments, the polypeptide inhibitsTGFβ1 with an IC50 of less than 1.0 nM, as determined using a reportergene assay. In some embodiments, the polypeptide inhibits TGFβ1 with anIC50 of less than 0.25 nM, as determined using a reporter gene assay. Insome embodiments, the polypeptide inhibits TGFβ1 with an IC50 of lessthan 0.1 nM, as determined using a reporter gene assay. In someembodiments, the polypeptide inhibits TGFβ1 with an IC50 of less than0.05 nM, as determined using a reporter gene assay. In some embodiments,the polypeptide inhibits TGFβ3 with an IC50 of less than 0.3 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.1 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.05 nM, asdetermined using a reporter gene assay. In some embodiments, thepolypeptide inhibits TGFβ3 with an IC50 of less than 0.04 nM, asdetermined using a reporter gene assay. In some embodiments, thereporter gene assay is a CAGA reporter assay.

In some embodiments, the disclosure provides for a homodimer comprisingany two of the polypeptides disclosed herein.

In some embodiments, the disclosure provides for an isolatedpolynucleotide comprising a coding sequence for any of the polypeptidesdisclosed herein. In some embodiments, the disclosure provides for arecombinant polynucleotide comprising a promoter sequence operablylinked to any of the polynucleotides disclosed herein. In someembodiments, the polynucleotide comprises a nucleotide sequence that isat least 80%, 85%, 90%, 95%, 97% or 100% identical to any one of SEQ IDNOs: 10, 12 or 14.

In some embodiments, the disclosure provides for a cell transformed withany of the polynucleotides disclosed herein. In some embodiments, cellis a mammalian cell. In some embodiments, the cell is a CHO cell or ahuman cell.

In some embodiments, the disclosure provides for a pharmaceuticalpreparation comprising pharmaceutically acceptable excipient and any ofthe polypeptides disclosed herein or any of the homodimers disclosedherein.

In some embodiments, the disclosure provides for a method of modulatingthe response of a cell to a TGFβ superfamily member, the methodcomprising exposing the cell to any of the polypeptides disclosed hereinor any of the homodimers disclosed herein. In some embodiments, thedisclosure provides for a method of treating a disease or conditionassociated with a TGFβ superfamily member in a patient in need thereof,the method comprising administering to the patient an effective amountof any of the polypeptides disclosed herein or any of the homodimersdisclosed herein. In some embodiments, the TGFβ superfamily member isTGFβ1 or TGFβ3. In some embodiments, the disease or condition is acancer. In some embodiments, the cancer is selected from stomach cancer,intestinal cancer, skin cancer, breast cancer, melanoma, bone cancer andthyroid cancer. In some embodiments, the disease or condition is afibrotic or sclerotic disease or condition. In some embodiments, thefibrotic or sclerotic disease or condition is selected from scleroderma,lupus erythematosis, pulmonary fibrosis, atherosclerosis, liverfibrosis, diffuse systemic sclerosis, glomerulonephritis, neuralscarring, dermal scarring, radiation-induced fibrosis, hepatic fibrosis,idiopathic pulmonary fibrosis and myelofibrosis. In some embodiments,the disease or condition is myelofibrosis. In some embodiments, thedisease or condition is selected from the group consisting of primarymyelofibrosis, post-polycythemia vera myelofibrosis, and post-essentialthrombocythemia myelofibrosis. In some embodiments, the disease orcondition is selected from the group consisting of low risk,intermediate-1 risk, intermediate-2 risk, or high-risk myelofibrosisaccording to the International Prognostic Scoring System (IPSS) or tothe to the dynamic IPSS (DIPSS). In some embodiments, the disease orcondition is heart disease. In some embodiments, the disease orcondition is selected from hereditary hemorrhagic telangiectasia (HHT),Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysmsyndrome, arterial tortuosity syndrome, pre-eclampsia, atherosclerosis,restenosis, and hypertrophic cardiomyopathy/congestive heart failure. Insome embodiments, the disease or condition is pulmonary hypertension. Insome embodiments, the pulmonary hypertension is Class I, Class II, ClassIII, or Class IV pulmonary hypertension as recognized by the WorldHealth Organization. In some embodiments, the disease or condition is akidney-associated disease or condition. In some embodiments, thekidney-associated disease or condition is selected from the groupconsisting of: chronic kidney diseases (or failure), acute kidneydiseases (or failure), primary kidney diseases, non-diabetic kidneydiseases, glomerulonephritis, interstitial nephritis, diabetic kidneydiseases, diabetic nephropathy, glomerulosclerosis, rapid progressiveglomerulonephritis, renal fibrosis, Alport syndrome, IDDM nephritis,mesangial proliferative glomerulonephritis, membranoproliferativeglomerulonephritis, crescentic glomerulonephritis, renal interstitialfibrosis, focal segmental glomerulosclerosis, membranous nephropathy,minimal change disease, pauci-immune rapid progressiveglomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent'sdisease, nephrocytinosis, Heymann nephritis, autosomal dominant (adult)polycystic kidney disease, autosomal recessive (childhood) polycystickidney disease, acute kidney injury, nephrotic syndrome, renal ischemia,podocyte diseases or disorders, proteinuria, glomerular diseases,membranous glomerulonephritis, focal segmental glomerulonephritis,pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,benign orthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, acute interstitial nephritis, establishedchronic kidney disease, renal artery stenosis, ischemic nephropathy,uremia, drug and toxin-induced chronic tubulointerstitial nephritis,reflux nephropathy, kidney stones, Goodpasture's syndrome, normocyticnormochromic anemia, renal anemia, diabetic chronic kidney disease,IgG4-related disease, von Hippel-Lindau syndrome, tuberous sclerosis,nephronophthisis, medullary cystic kidney disease, renal cell carcinoma,adenocarcinoma, nephroblastoma, lymphoma, leukemia, hyposialylationdisorder, chronic cyclosporine nephropathy, renal reperfusion injury,renal dysplasia, azotemia, bilateral arterial occlusion, acute uric acidnephropathy, hypovolemia, acute bilateral obstructive uropathy,hypercalcemic nephropathy, hemolytic uremic syndrome, acute urinaryretention, malignant nephrosclerosis, postpartum glomerulosclerosis,scleroderma, non-Goodpasture's anti-GBM disease, microscopicpolyarteritis nodosa, allergic granulomatosis, acute radiationnephritis, post-streptococcal glomerulonephritis, Waldenstrom'smacroglobulinemia, analgesic nephropathy, arteriovenous fistula,arteriovenous graft, dialysis, ectopic kidney, medullary sponge kidney,renal osteodystrophy, solitary kidney, hydronephrosis, microalbuminuria,uremia, haematuria, hyperlipidemia, hypoalbuminaemia, lipiduria,acidosis, hyperkalemia, and edema. In some embodiments, thekidney-associated disease or condition is chronic kidney disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of native precursor for the B(short) isoform of human TGFβ receptor type II (hTβRII) (NP_003233.4).Solid underline indicates the mature extracellular domain (ECD)(residues 23-159), and double underline indicates valine that isreplaced in the A (long) isoform. Dotted underline denotes leader(residues 1-22).

FIG. 2 shows the amino acid sequence of native precursor for the A(long) isoform of human TβRII (NP 001020018.1). Solid underlineindicates the mature ECD (residues 23-184), and double underlineindicates the splice-generated isoleucine substitution. Dotted underlinedenotes leader (residues 1-22).

FIG. 3 shows a comparison of the linker sequences of five differentTβRII constructs (SEQ ID NOS 62-66, respectively, in order ofappearance).

FIGS. 4A and 4B show in tabular form the binding affinity between TGFβ1and TGFβ3 and one of several different TβRII-Fc fusion proteinconstructs.

FIGS. 5A and 5C graph the results from reporter gene assays testing theaffinity of TGFβ1 for one of several different TβRII-Fc fusion proteinconstructs. FIGS. 5B and 5D graph the results from reporter gene assaystesting the affinity of the TGFβ3 for one of several different TβRII-Fcfusion protein constructs. FIGS. 5E and 5F provide ICso data from thesesame experiments in tabular form.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

Proteins described herein are the human forms, unless otherwisespecified. NCBI references for the proteins are as follows: human TβRIIisoform A (hTβRII_(long)), NP_001020018.1 and human TβRII isoform B(hTβRII_(short)), (NP_003233.4). Sequences of native TβRII proteins fromhuman are set forth in FIGS. 1 and 2. In some embodiments, the TβRIIproteins are from non-human animals, such as a mouse, rat, cow ormonkey.

The TGFβ superfamily contains a variety of growth factors that sharecommon sequence elements and structural motifs. These proteins are knownto exert biological effects on a large variety of cell types in bothvertebrates and invertebrates. Members of the superfamily performimportant functions during embryonic development in pattern formationand tissue specification and can influence a variety of differentiationprocesses, including adipogenesis, myogenesis, chondrogenesis,cardiogenesis, hematopoiesis, neurogenesis, and epithelial celldifferentiation. By manipulating the activity of a member of the TGFβfamily, it is often possible to cause significant physiological changesin an organism. For example, the Piedmontese and Belgian Blue cattlebreeds carry a loss-of-function mutation in the GDF8 (also calledmyostatin) gene that causes a marked increase in muscle mass. Grobet etal., Nat Genet. 1997, 17(1):71-4. Similarly, in humans, inactive allelesof GDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

TGFβ signals are mediated by heteromeric complexes of type I (e.g. TOROand type II (e.g. TβRII) serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins upon ligandstimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. 1:169-178). Thesetype I and type II receptors are transmembrane proteins, composed of aligand-binding extracellular domain with cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine specificity. Type I receptors are essential forsignaling; and type II receptors are required for binding ligands andfor expression of type I receptors. Type I and II receptors form astable complex after ligand binding, resulting in phosphorylation oftype I receptors by type II receptors. TGFβ has three mammalianisoforms, TGFβ1, TGFβ2 and TGFβ3, each with distinct functions in vivo.The binding of TGFβs to TβRII is a crucial step in initiating activationof the TGFβ signaling pathway, leading to phosphorylation of SMAD2, andtranslocation of the activated SMAD2/SMAD4 complex to the nucleus tomodulate gene expression.

Thus, in certain aspects, the disclosure provides TβRII polypeptides asantagonists of TGFβ1 or TGFβ3 for use in treating various TGFβ1- orTGFβ3-associated disorders. While not wishing to be bound to anyparticular mechanism of action, it is expected that such polypeptidesact by binding to TGFβ1 or TGFβ3 and inhibiting the ability of theseligands to form ternary signaling complexes.

The disclosure provides for fusion proteins comprising TβRIIpolypeptides and a heterologous portion (e.g., an Fc portion). Inparticular embodiments, the TβRII portion and the heterologous portionare fused by means of a linker. As described in greater detail below,the disclosure demonstrates that TβRII-Fc fusion proteins comprisinglinkers of certain lengths (e.g., a linker having 21 amino acids) weresurprisingly able to bind TGFβ-1 and TGFβ-3 with stronger affinity thanTβRII-Fc fusion proteins having a linker of only four amino acids.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. The term “sequencesimilarity,” in all its grammatical forms, refers to the degree ofidentity or correspondence between nucleic acid or amino acid sequencesthat may or may not share a common evolutionary origin. However, incommon usage and in the instant application, the term “homologous,” whenmodified with an adverb such as “highly,” may refer to sequencesimilarity and may or may not relate to a common evolutionary origin.

“Percent (%) sequence identity” or “percent (%) identical” with respectto a reference polypeptide (or nucleotide) sequence is defined as thepercentage of amino acid residues (or nucleic acids) in a candidatesequence that are identical to the amino acid residues (or nucleicacids) in the reference polypeptide (nucleotide) sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid (nucleic acid) sequence identity values are generated using thesequence comparison computer program ALIGN-2. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc., and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is publiclyavailable from Genentech, Inc., South San Francisco, Calif., or may becompiled from the source code. The ALIGN-2 program should be compiledfor use on a UNIX operating system, including digital UNIX V4.0D. Allsequence comparison parameters are set by the ALIGN-2 program and do notvary.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers. As used herein, the term “comprises”also encompasses the use of the narrower terms “consisting” and“consisting essentially of” The term “consisting essentially of” islimited to the specified materials or steps and those that do notmaterially affect the basic and novel characteristics of theinvention(s) disclosed herein.

The term “appreciable affinity” as used herein means binding with adissociation constant (K_(D)) of less than 50 nM.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. The chain may be linear or branched, it may comprise modifiedamino acids, and/or may be interrupted by non-amino acids. The termsalso encompass an amino acid chain that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that the polypeptides can occur as single chains orassociated chains.

2. TβRII Polypeptides

Naturally occurring TβRII proteins are transmembrane proteins, with aportion of the protein positioned outside the cell (the extracellularportion) and a portion of the protein positioned inside the cell (theintracellular portion). Aspects of the present disclosure encompassvariant TβRII polypeptides comprising mutations within the extracellulardomain and/or truncated portions of the extracellular domain of TβRII.As described above, human TβRII occurs naturally in at least twoisoforms—A (long) and B (short)—generated by alternative splicing in theextracellular domain (ECD) (FIGS. 1 and 2 and SEQ ID NOS: 1 and 2). SEQID NO: 27, which corresponds to residues 23-159 of SEQ ID NO: 1, depictsthe native full-length extracellular domain of the short isoform ofTβRII. SEQ ID NO: 18, which corresponds to residues 23-184 of SEQ ID NO:2, depicts the native full-length extracellular domain of the longisoform of TβRII. Unless noted otherwise, amino acid position numberingwith regard to variants based on the TβRII short and long isoformsrefers to the corresponding position in the native precursors, SEQ IDNO: 1 and SEQ ID NO: 2, respectively.

In certain embodiments, the disclosure provides variant TβRIIpolypeptides. A TβRII polypeptide of the disclosure may bind to andinhibit the function of a TGFβ superfamily member, such as but notlimited to, TGFβ1 or TGFβ3. TβRII polypeptides may include a polypeptideconsisting of, or comprising, an amino acid sequence at least 80%identical, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to a truncated ECD domain of a naturally occurring TβRIIpolypeptide, whose C-terminus occurs at any of amino acids 153-159 ofSEQ ID NO: 1. TβRII polypeptides may include a polypeptide consistingof, or comprising, an amino acid sequence at least 80% identical, andoptionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto a truncated ECD domain of a naturally occurring TβRII polypeptide,whose C-terminus occurs at any of amino acids 178-184 of SEQ ID NO: 2.In particular embodiments, the TβRII polypeptides comprise an amino acidsequence at least 80% identical, and optionally at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQID NO: 18. Optionally, a TβRII polypeptide does not include more than 5consecutive amino acids, or more than 10, 20, 30, 40, 50, 52, 60, 70,80, 90, 100, 150 or 200 or more consecutive amino acids from a sequenceconsisting of amino acids 160-567 of SEQ ID NO: 1 or from a sequenceconsisting of amino acids 185-592 of SEQ ID NO: 2. In some embodiments,the TβRII polypeptide does not include amino acids 160-567 of SEQ IDNO: 1. In some embodiments, the TβRII polypeptide does not include aminoacids 1-22 of SEQ ID NO: 1. In some embodiments, the TβRII polypeptidedoes not include amino acids 1-22 and 160-567 of SEQ ID NO: 1. In someembodiments, the TβRII polypeptide does not include amino acids 185-592of SEQ ID NO: 2. In some embodiments, the TβRII polypeptide does notinclude amino acids 1-22 of SEQ ID NO: 2. In some embodiments, the TβRIIpolypeptide does not include amino acids 1-22 and 185-592 of SEQ ID NO:2. The unprocessed TβRII polypeptide may either include or exclude anysignal sequence, as well as any sequence N-terminal to the signalsequence. As elaborated herein, the N-terminus of the mature (processed)TβRII polypeptide may occur at any of amino acids 23-35 of SEQ ID NO: 1or 23-60 of SEQ ID NO: 2. Examples of mature TβRII polypeptides include,but are not limited to, amino acids 23-159 of SEQ ID NO: 1 (set forth inSEQ ID NO: 27), amino acids 29-159 of SEQ ID NO: 1 (set forth in SEQ IDNO: 28), amino acids 35-159 of SEQ ID NO: 1 (set forth in SEQ ID NO:29), amino acids 23-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 30),amino acids 29-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 31), aminoacids 35-153 of SEQ ID NO: 1 (set forth in SEQ ID NO: 32), amino acids23-184 of SEQ ID NO: 2 (set forth in SEQ ID NO: 18), amino acids 29-184of SEQ ID NO: 2 (set forth in SEQ ID NO: 33), amino acids 60-184 of SEQID NO: 2 (set forth in SEQ ID NO: 29), amino acids 23-178 of SEQ ID NO:2 (set forth in SEQ ID NO: 34), amino acids 29-178 of SEQ ID NO: 2 (setforth in SEQ ID NO: 35), and amino acids 60-178 of SEQ ID NO: 2 (setforth in SEQ ID NO: 32). It will be understood by one of skill in theart that corresponding variants based on the long isoform of TβRII willinclude nucleotide sequences encoding the 25-amino acid insertion alongwith a conservative Val-Ile substitution at the flanking positionC-terminal to the insertion. The TβRII polypeptides accordingly mayinclude isolated extracellular portions of TβRII polypeptides, includingboth the short and the long isoforms, variants thereof (includingvariants that comprise, for example, no more than 2, 3, 4, 5, 10, 15,20, 25, 30, or 35 amino acid substitutions in the sequence correspondingto amino acids 23-159 of SEQ ID NO: 1 or amino acids 23-184 of SEQ IDNO: 2), fragments thereof, and fusion proteins comprising any of theforegoing, but in each case preferably any of the foregoing TβRIIpolypeptides will retain substantial affinity for at least one of, orboth of, TGFβ1 or TGFβ3. Generally, a TβRII polypeptide will be designedto be soluble in aqueous solutions at biologically relevanttemperatures, pH levels, and osmolarity.

In some embodiments, the variant TβRII polypeptides of the disclosurecomprise one or more mutations in the extracellular domain that conferan altered ligand binding profile. A TβRII polypeptide may include one,two, five or more alterations in the amino acid sequence relative to thecorresponding portion of a naturally occurring TβRII polypeptide. Insome embodiments, the mutation results in a substitution, insertion, ordeletion at the position corresponding to position 70 of SEQ ID NO: 1.In some embodiments, the mutation results in a substitution, insertion,or deletion at the position corresponding to position 110 of SEQ IDNO: 1. Examples include, but are not limited to, an N to D substitutionor a D to K substitution in the positions corresponding to positions 70and 110, respectively, of SEQ ID NO: 1. Examples of such variant TβRIIpolypeptides include, but are not limited to, the sequences set forth inSEQ ID NOs: 36-39. A TβRII polypeptide may comprise a polypeptide orportion thereof that is encoded by any one of SEQ ID NOs: 10, 12, 14 or16, or silent variants thereof or nucleic acids that hybridize to thecomplement thereof under stringent hybridization conditions. Inparticular embodiments, a TβRII polypeptide may comprise a polypeptideor portion thereof that is encoded by any one of SEQ ID NO: 12, orsilent variants thereof or nucleic acids that hybridize to thecomplement thereof under stringent hybridization conditions.

In some embodiments, the variant TβRII polypeptides of the disclosurefurther comprise an insertion of 36 amino acids (SEQ ID NO: 41) betweenthe pair of glutamate residues (positions 151 and 152 of SEQ ID NO: 1,or positions 176 and 177 of SEQ ID NO: 2) located near the C-terminus ofthe human TβRII ECD, as occurs naturally in the human TβRII isoform C(Konrad et al., BMC Genomics 8:318, 2007).

The disclosure further demonstrates that TβRII polypeptides can bemodified to selectively antagonize TβRII ligands. The N70 residuerepresents a potential glycosylation site. In some embodiments, theTβRII polypeptides are aglycosylated. In some embodiments, the TβRIIpolypeptides are aglycosylated or have reduced glycosylation at positionAsn157. In some embodiments, the TβRII polypeptides are aglycosylated orhave reduced glycosylation at position Asn73.

In certain embodiments, a TβRII polypeptide binds to TGFβ1, and theTβRII polypeptide does not show substantial binding to TGFβ3. In certainembodiments, a TβRII polypeptide binds to TGFβ3, and the TβRIIpolypeptide does not show substantial binding to TGFβ1. Binding may beassessed using purified proteins in solution or in a surface plasmonresonance system, such as a Biacore™ system.

In certain embodiments, a TβRII polypeptide inhibits TGFβ1 cellularsignaling, and the TβRII polypeptide has an intermediate or limitedinhibitory effect on TGFβ3 signaling. In certain embodiments, a TβRIIpolypeptide inhibits TGFβ3 cellular signaling, and the TβRII polypeptidehas an intermediate or limited inhibitory effect on TGFβ1 signaling.Inhibitory effect on cell signaling can be assayed by methods known inthe art.

Taken together, an active portion of a TβRII polypeptide may compriseamino acid sequences 23-153, 23-154, 23-155, 23-156, 23-157, or 23-158of SEQ ID NO: 1, as well as variants of these sequences starting at anyof amino acids 24-35 of SEQ ID NO: 1. Similarly, an active portion of aTβRII polypeptide may comprise amino acid sequences 23-178, 23-179,23-180, 23-181, 23-182, or 23-183 of SEQ ID NO: 2, as well as variantsof these sequences starting at any of amino acids 24-60 of SEQ ID NO: 2.Exemplary TβRII polypeptides comprise amino acid sequences 29-159,35-159, 23-153, 29-153 and 35-153 of SEQ ID NO: 1 or amino acidsequences 29-184, 60-184, 23-178, 29-178 and 60-178 of SEQ ID NO: 2.Variants within these ranges are also contemplated, particularly thosehaving at least 80%, 85%, 90%, 95%, or 99% identity to the correspondingportion of SEQ ID NO: 1 or SEQ ID NO: 2. A TβRII polypeptide may beselected that does not include the sequence consisting of amino acids160-567 of SEQ ID NO: 1 or amino acids 185-592 of SEQ ID NO: 2. Inparticular embodiments, the TβRII polypeptides comprise an amino acidsequence at least 80% identical, and optionally at least 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQID NO: 18.

As described above, the disclosure provides TβRII polypeptides sharing aspecified degree of sequence identity or similarity to a naturallyoccurring TβRII polypeptide. To determine the percent identity of twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The amino acid residues at corresponding amino acid positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue as the corresponding position in the second sequence,then the molecules are identical at that position (as used herein aminoacid “identity” is equivalent to amino acid “homology”). The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

In one embodiment, the percent identity between two amino acid sequencesis determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package. In a specific embodiment, the followingparameters are used in the GAP program: either a Blosum 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)). Exemplary parameters include usinga NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. Unless otherwise specified,percent identity between two amino acid sequences is to be determinedusing the GAP program using a Blosum 62 matrix, a GAP weight of 10 and alength weight of 3, and if such algorithm cannot compute the desiredpercent identity, a suitable alternative disclosed herein should beselected.

In another embodiment, the percent identity between two amino acidsequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

Another embodiment for determining the best overall alignment betweentwo amino acid sequences can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.,6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is presented in terms of percent identity. In oneembodiment, amino acid sequence identity is performed using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci., 6:237-245 (1990)). In a specific embodiment, parametersemployed to calculate percent identity and similarity of an amino acidalignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5 and Gap Size Penalty=0.05.

TβRII polypeptides may additionally include any of various leadersequences at the N-terminus. Such a sequence would allow the peptides tobe expressed and targeted to the secretion pathway in a eukaryoticsystem. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783 (1992).Alternatively, a native TβRII signal sequence may be used to effectextrusion from the cell. Possible leader sequences include nativeleaders, tissue plasminogen activator (TPA) and honeybee mellitin (SEQID NOs. 22-24, respectively). Examples of TβRII-Fc fusion proteinsincorporating a TPA leader sequence include SEQ ID NOs: 11, 13, 15 and17. Processing of signal peptides may vary depending on the leadersequence chosen, the cell type used and culture conditions, among othervariables, and therefore actual N-terminal start sites for mature TβRIIpolypeptides may shift by 1, 2, 3, 4 or 5 amino acids in either theN-terminal or C-terminal direction. Examples of TβRII-Fc fusion proteinsinclude SEQ ID NOs: 11, 13, 15 and 17. It will be understood by one ofskill in the art that corresponding variants based on the long isoformof TβRII will include the 25-amino acid insertion along with aconservative Val-Ile substitution at the flanking position C-terminal tothe insertion.

In some embodiments, any of the TβRII polypeptides disclosed herein areat least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to theamino acid sequence of any one of SEQ ID NOs: 18, 27, 30, 34, 36, 37,38, 39, 48, 49 or 51, but lack one or more N-terminal amino acids ascompared to the amino acid sequences of SEQ ID NO: 18, 27, 30, 34, 36,37, 38, 39, 48, 49 or 51. In some embodiments, the TβRII polypeptidelacks the amino acid corresponding to the first amino acid (threonine)of any one of SEQ ID NOs: 18, 27, 30, 34, 36, 37, 38, 39, 48, 49 or 51.In some embodiments, the TβRII polypeptide lacks the amino acidscorresponding to the first and second amino acids (threonine andisoleucine, respectively) of any one of SEQ ID NOs: 18, 27, 30, 34, 36,37, 38, 39, 48, 49 or 51. In some embodiments, the TβRII polypeptidelacks the amino acids corresponding to the first, second and third aminoacids (threonine, isoleucine, and proline, respectively) of any one ofSEQ ID NOs: 18, 27, 30, 34, 36, 37, 38, 39, 48, 49 or 51. In someembodiments, the TβRII polypeptide lacks the amino acids correspondingto the first, second, third and fourth amino acids (threonine,isoleucine, proline, proline, respectively) of any one of SEQ ID NOs:18, 27, 30, 34, 36, 37, 38, 39, 48, 49 or 51.

In some embodiments, any of the TβRII polypeptides disclosed herein areat least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to theamino acid sequence of any one of SEQ ID NOs: 18 or 51, but lack theamino acid corresponding to the first amino acid (threonine) of SEQ IDNO: 18 or 51. In some embodiments, the TβRII polypeptide lacks the aminoacids corresponding to the first and second amino acids (threonine andisoleucine, respectively) of SEQ ID NO: 18 or 51. In some embodiments,the TβRII polypeptide lacks the amino acids corresponding to the first,second and third amino acids (threonine, isoleucine, and proline,respectively) of SEQ ID NO: 18 or 51. In some embodiments, the TβRIIpolypeptide lacks the amino acids corresponding to the first, second,third and fourth amino acids (threonine, isoleucine, proline, proline,respectively) of SEQ ID NO: 18 or 51.

In some embodiments, the disclosure provides for a compositioncomprising a mixture of TβRII polypeptides, wherein the TβRIIpolypeptides in the composition each comprise an amino acid sequencethat is at least 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100%identical to the amino acid sequence of any one of SEQ ID NOs: 18, 27,30, 34, 36, 37, 38, 39, 48, 49 or 51; but wherein at least a portion ofthe TβRII polypeptides (e.g., at least 1%, 3%, 4%, 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) in the composition includethe amino acids corresponding to the first, second, third and fourthamino acids (threonine, isoleucine, proline and proline, respectively)of any one of SEQ ID NOs: 18, 27, 30, 34, 36, 37, 38, 39, 48, 49 or 51;and wherein at least a portion of the TβRII polypeptides (e.g., at least1%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%) in the composition lack one or more of the amino acidscorresponding to the first, second, third and fourth amino acids(threonine, isoleucine, proline and proline, respectively) of any one ofSEQ ID NOs: 18, 27, 30, 34, 36, 37, 38, 39, 48, 49 or 51. In someembodiments, the disclosure provides for a composition comprising amixture of TβRII polypeptides, wherein the TβRII polypeptides are atleast 80%, 85%, 90%, 92%, 94%, 95%, 97%, 99% or 100% identical to theamino acid sequence of any one of SEQ ID NOs: 18 or 51, but wherein atleast 30% to 80% of the TβRII polypeptides in the composition lack theamino acid corresponding to the first amino acid (threonine) of SEQ IDNO: 18 or 51.

In certain embodiments, the present disclosure contemplates specificmutations of the TβRII polypeptides so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (orasparagine-X-serine) (where “X” is any amino acid) which is specificallyrecognized by appropriate cellular glycosylation enzymes. The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the wild-type TβRIIpolypeptide (for O-linked glycosylation sites). A variety of amino acidsubstitutions or deletions at one or both of the first or third aminoacid positions of a glycosylation recognition site (and/or amino aciddeletion at the second position) results in non-glycosylation at themodified tripeptide sequence. Another means of increasing the number ofcarbohydrate moieties on a TβRII polypeptide is by chemical or enzymaticcoupling of glycosides to the TβRII polypeptide. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine; (b) free carboxyl groups; (c) free sulfhydryl groups such asthose of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein. Removal of one or morecarbohydrate moieties present on a TβRII polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the TβRII polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on TβRII polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of a TβRII polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, TβRII polypeptides for use inhumans will be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines, yeast cell lines with engineeredglycosylation enzymes, and insect cells are expected to be useful aswell.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of a TβRII polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, TβRII polypeptide variants which can act as either agonistsor antagonist, or alternatively, which possess novel activities alltogether. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, a TβRIIpolypeptide variant may be screened for ability to bind to a TβRIIligand, to prevent binding of a TβRII ligand to a TβRII polypeptide orto interfere with signaling caused by a TβRII ligand. The activity of aTβRII polypeptide or its variants may also be tested in a cell-based orin vivo assay, particularly any of the assays disclosed in the Examples.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a TβRII polypeptidecomprising an extracellular domain of a naturally occurring TβRIIpolypeptide. Likewise, mutagenesis can give rise to variants which haveserum half-lives dramatically different than the corresponding wild-typeTβRII polypeptide. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or otherprocesses which result in destruction of, or otherwise elimination orinactivation of, a native TβRII polypeptide. Such variants, and thegenes which encode them, can be utilized to alter TβRII polypeptidelevels by modulating the half-life of the TβRII polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and can allow tighter control of recombinant TβRII polypeptidelevels within the patient. In an Fc fusion protein, mutations may bemade in the linker (if any) and/or the Fc portion to alter the half-lifeof the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential TβRII polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential TβRIIpolypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential TβRII polypeptidevariants can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then be ligatedinto an appropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura etal., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al., (1990) Science249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin etal., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, TβRII polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of TβRII polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of TβRII polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include TβRII ligand binding assays and ligand-mediated cellsignaling assays.

In certain embodiments, the TβRII polypeptides of the disclosure mayfurther comprise post-translational modifications in addition to anythat are naturally present in the TβRII polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, pegylation (polyethyleneglycol) and acylation. As a result, the modified TβRII polypeptides maycontain non-amino acid elements, such as polyethylene glycols, lipids,mono- or poly-saccharides, and phosphates. Effects of such non-aminoacid elements on the functionality of a TβRII polypeptide may be testedas described herein for other TβRII polypeptide variants. When a TβRIIpolypeptide is produced in cells by cleaving a nascent form of the TβRIIpolypeptide, post-translational processing may also be important forcorrect folding and/or function of the protein. Different cells (such asCHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK-293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the TβRII polypeptides.

3. Linkers

The disclosure provides for TβRII fusion proteins, and in theseembodiments, the TβRII portion is connected to the heterologous portion(e.g., Fc portion) by means of a linker. In some embodiments, thelinkers are glycine and serine rich linkers. Other near neutral aminoacids, such as, but not limited to, Thr, Asn, Pro and Ala, may also beused in the linker sequence. In some embodiments, the linker comprisesvarious permutations of amino acid sequences containing Gly and Ser. Insome embodiments, the linker is greater than 10 amino acids in length.In further embodiments, the linkers have a length of at least 12, 15,20, 21, 25, 30, 35, 40, 45 or 50 amino acids. In some embodiments, thelinker is less than 40, 35, 30, 25, 22 or 20 amino acids. In someembodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-21, 10-15, 10,15-25, 17-22, 20, or 21 amino acids in length. In preferred embodiments,the linker comprises the amino acid sequence GlyGlyGlyGlySer (GGGGS)(SEQ ID NO: 19), or repetitions thereof (GGGGS)n, where n≥2 (SEQ ID NO:57). In particular embodiments n≥3, or n=3-10. The application teachesthe surprising finding that proteins comprising a TβRII portion and aheterologous portion fused together by means of a (GGGGS)4 linker (SEQID NO: 58) were associated with a stronger affinity for TGFβ1 and TGFβ3as compared to a TβRII fusion protein where n<4. As such, in preferredembodiments, n≥4, or n=4-10. The application also teaches that proteinscomprising (GGGGS)n linkers (‘GGGGS’ disclosed as SEQ ID NO: 19) inwhich n>4 had similar inhibitory properties as proteins having the(GGGGS)4 linker (SEQ ID NO: 58). As such, in some embodiments, n is notgreater than 4 in a (GGGGS)n linker (SEQ ID NO: 19). In someembodiments, n=4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, or 5-6. In someembodiments, n=3, 4, 5, 6, or 7. In particular embodiments, n=4. In someembodiments, a linker comprising a (GGGGS)_(n) sequence (SEQ ID NO: 19)also comprises an N-terminal threonine. In some embodiments, the linkeris any one of the following:

(SEQ ID NO: 21) GGGGSGGGGS (SEQ ID NO: 4) TGGGGSGGGGS (SEQ ID NO: 5)TGGGGSGGGGSGGGGS (SEQ ID NO: 6) TGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 25)TGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 26)TGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS or (SEQ ID NO: 40)TGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.In some embodiments, the linker comprises the amino acid sequence ofTGGGPKSCDK (SEQ ID NO: 7). In some embodiments, the linker is any one ofSEQ ID NOs: 21, 4-7, 25-26 or 40 lacking the N-terminal threonine. Insome embodiments, the linker does not comprise the amino acid sequenceof SEQ ID NO: 26 or 40.

4. Heterologous Portions

In certain aspects, functional variants or modified forms of the TβRIIpolypeptides include fusion proteins having at least a portion of theTβRII polypeptides and one or more heterologous portions. Well-knownexamples of such heterologous portions include, but are not limited to,polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. Aheterologous portion may be selected so as to confer a desired property.For example, some heterologous portions are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆ (SEQ ID NO: 61)) fusionpartners. As another example, a heterologous portion may be selected soas to facilitate detection of the TβRII polypeptides. Examples of suchdetection domains include the various fluorescent proteins (e.g., GFP)as well as “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, theheterologous portions have a protease cleavage site, such as for FactorXa or Thrombin, which allows the relevant protease to partially digestthe fusion proteins and thereby liberate the recombinant proteinstherefrom. The liberated proteins can then be isolated from theheterologous portion by subsequent chromatographic separation. Incertain preferred embodiments, a TβRII polypeptide is fused with adomain that stabilizes the TβRII polypeptide in vivo (a “stabilizer”domain). By “stabilizing” is meant anything that increases serum halflife, regardless of whether this is because of decreased destruction,decreased clearance by the kidney, or other pharmacokinetic effect.Fusions with the Fc portion of an immunoglobulin are known to conferdesirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of heterologous portions that may be selectedinclude multimerizing (e.g., dimerizing, tetramerizing) domains andfunctional domains.

As specific examples, the present disclosure provides fusion proteinscomprising variants of TβRII polypeptides fused to an Fc domain sequenceof SEQ ID NO: 20. Optionally, the Fc domain has one or more mutations atresidues such as Asp-265, Lys-322, and Asn-434 (numbered in accordancewith the corresponding full-length IgG). In certain cases, the mutant Fcdomain having one or more of these mutations (e.g., Asp-265 mutation)has reduced ability of binding to the Fcγ receptor relative to awildtype Fc domain. In other cases, the mutant Fc domain having one ormore of these mutations (e.g., Asn-434 mutation) has increased abilityof binding to the MHC class I-related Fc-receptor (FcRN) relative to awildtype Fc domain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a TβRII polypeptide may be placed C-terminalto a heterologous domain, or, alternatively, a heterologous domain maybe placed C-terminal to a TβRII polypeptide. The TβRII polypeptidedomain and the heterologous domain need not be adjacent in a fusionprotein, and additional domains or amino acid sequences may be includedC- or N-terminal to either domain or between the domains.

As used herein, the term “immunoglobulin Fc domain” or simply “Fc” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2domain and a CH3 domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain. In some embodiments, the immunoglobulin Fc region is a humanimmunoglobulin Fc region.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). Other classes of immunoglobulin, IgA (Iga), IgD (Igδ), IgE (IgE) andIgM (Igp), may be used. The choice of appropriate immunoglobulin heavychain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087and 5,726,044. The choice of particular immunoglobulin heavy chainconstant region sequences from certain immunoglobulin classes andsubclasses to achieve a particular result is considered to be within thelevel of skill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH₃ domain of Fcgamma or the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the methods and compositions disclosed herein.One example would be to introduce amino acid substitutions in the upperCH2 region to create an Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. Immunol. 159:3613).

In some embodiments, the disclosure provides for TβRII polypeptidesfusion proteins comprising an amino acid sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of any one of SEQ ID NOs: 11, 13, 15 and 17, or biologicallyactive fragments thereof. In some embodiments, the TβRII polypeptidesfusion proteins comprise an amino acid sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of any one of SEQ ID NOs: 11, 13, and 15, or biologicallyactive fragments thereof. In some embodiments, the TβRII polypeptidesfusion proteins comprise an amino acid sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of any one of SEQ ID NO: 13, or a biologically active fragmentthereof. In some embodiments, the TβRII polypeptides fusion proteinscomprise an amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of any one ofSEQ ID NO: 50, or a biologically active fragment thereof. In someembodiments, the TβRII polypeptides fusion proteins comprise an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NO: 51, or abiologically active fragment thereof. In some embodiments, the TβRIIpolypeptides fusion proteins comprise an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence of any one of SEQ ID NO: 52, or a biologically activefragment thereof. In some embodiments, the TβRII polypeptides fusionproteins comprise an amino acid sequence that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anyone of SEQ ID NO: 53, or a biologically active fragment thereof. In someembodiments, the TβRII polypeptides fusion proteins comprise an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of any one of SEQ ID NO: 54, or abiologically active fragment thereof. In some embodiments, the TβRIIpolypeptides fusion proteins comprise an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence of any one of SEQ ID NO: 55, or a biologically activefragment thereof. In some embodiments, the TβRII polypeptides fusionproteins comprise an amino acid sequence that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anyone of SEQ ID NO: 56, or a biologically active fragment thereof. In someembodiments, the TβRII polypeptides fusion protein comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 20, or a biologicallyactive fragment thereof.

In some embodiments, the fusion proteins described herein have improvedbinding affinity for TGFβ1 and TGFβ3. In some embodiments, a fusionprotein comprising a linker at least 10 amino acids in length (e.g., afusion protein having the amino acid sequence of any one of SEQ ID NOs:11, 13, 15, and 50-56) has improved binding affinity for TGFβ1 and TGFβ3as compared to a reference fusion protein (e.g., a fusion protein havingthe amino acid sequence of SEQ ID NO: 9). In some embodiments, thefusion protein binds to TGFβ1 with a K_(D) of less than 200 pM, lessthan 150 pM, less than 100 pM, less than 75 pM, less than 50 pM or lessthan 25 pM. In some embodiments, the fusion protein binds to TGFβ3 witha K_(D) of less than 75 pM, less than 70 pM, less than 60 pM, less than50 pM, less than 40 pM, less than 35 pM, less than 25 pM, less than 15,less than 10, or less than 5 pM.

In some embodiments any of the polypeptides disclosed herein inhibitsTGFβ1 and/or TGFβ3 in a measurable assay. In some embodiments, thepolypeptide inhibits TGFβ1 with an IC₅₀ of less than 1.0, 0.9, 0.8, 0.7,0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.08, 0.09, 0.07, 0.06, 0.05, 0.04, 0.03,or 0.02 nM, as determined using a reporter gene assay. In someembodiments, the polypeptide inhibits TGFβ3 with an IC₅₀ of less than1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07,0.06, 0.05, 0.04, 0.03, or 0.02 nM, as determined using a reporter geneassay. In some embodiments, the reporter gene assay is a CAGA reporterassay. In some embodiments, the CAGA assay is based on a human lungcarcinoma cell line transfected with a pGL3(CAGA)12 reporter plasmid(Dennler et al, 1998, EMBO 17: 3091-3100) as well as a Renilla reporterplasmid (pRLCMV) to control for transfection efficiency. The CAGA motifis present in the promoters of TGFβ-responsive genes (for example,PAI-1), so this vector is of general use for factors signaling throughSMAD2 and SMAD3. See, e.g., Example 2.

5. Fusion Polypeptides

In some embodiments, the disclosure provides for TβRII-containing fusionpolypeptides. The fusion polypeptides may be prepared according to anyof the methods disclosed herein or that are known in the art.

In some embodiments, any of the fusion polypeptides disclosed hereincomprises the following components: a) any of the TβRII polypeptidesdisclosed herein (“A”), b) any of the linkers disclosed herein (“B”), c)any of the heterologous portions disclosed herein (“C”), and optionallya linker (“X”). In such embodiments, the fusion polypeptide may bearranged in a manner as follows (N-terminus to C-terminus): A-B—C orC—B-A. In such embodiments, the fusion polypeptide may be arranged in amanner as follows (N-terminus to C-terminus): X-A-B—C or X—C—B-A. Insome embodiments, the fusion polypeptide comprises each of A, B and C(and optionally a leader sequence such as the amino acid sequence of SEQID NO: 23), and comprises no more than 100, 90, 80, 70, 60, 50, 40, 30,20, 10, 5, 4, 3, 2 or 1 additional amino acids (but which may includefurther post-translational modifications, such as PEGylation).

In some embodiments, the fusion polypeptide comprises a leader sequence(e.g., SEQ ID NO: 23) positioned in a manner as follows (N-terminus toC-terminus): X-A-B—C, and the fusion polypeptide comprises 1, 2, 3, 4,or 5 amino acids between X and A. In some embodiments, the fusionpolypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positionedin a manner as follows (N-terminus to C-terminus): X—C—B-A, and thefusion polypeptide comprises 1, 2, 3, 4, or 5 amino acids between X andC. In some embodiments, the fusion polypeptide comprises a leadersequence (e.g., SEQ ID NO: 23) positioned in a manner as follows(N-terminus to C-terminus): X-A-B—C, and the fusion polypeptidecomprises an alanine between X and A. In some embodiments, the fusionpolypeptide comprises a leader sequence (e.g., SEQ ID NO: 23) positionedin a manner as follows (N-terminus to C-terminus): X—C—B-A, and thefusion polypeptide comprises an alanine between X and C. In someembodiments, the fusion polypeptide comprises a leader sequence (e.g.,SEQ ID NO: 23) positioned in a manner as follows (N-terminus toC-terminus): X-A-B—C, and the fusion polypeptide comprises a glycine andan alanine between X and A. In some embodiments, the fusion polypeptidecomprises a leader sequence (e.g., SEQ ID NO: 23) positioned in a manneras follows (N-terminus to C-terminus): X—C—B-A, and the fusionpolypeptide comprises a glycine and an alanine between X and C. In someembodiments, the fusion polypeptide comprises a leader sequence (e.g.,SEQ ID NO: 23) positioned in a manner as follows (N-terminus toC-terminus): X-A-B—C, and the fusion polypeptide comprises a threoninebetween X and A. In some embodiments, the fusion polypeptide comprises aleader sequence (e.g., SEQ ID NO: 23) positioned in a manner as follows(N-terminus to C-terminus): X—C—B-A, and the fusion polypeptidecomprises a threonine between X and C.

In some embodiments, the fusion polypeptide comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, or 99% identical to any ofthe TβRII polypeptide amino acid sequences disclosed herein (e.g., SEQID NO: 18), wherein the TβRII polypeptide portion of the fusionpolypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation).

In some embodiments, the fusion polypeptide comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, or 99% identical to any ofthe linker sequences disclosed herein (e.g., SEQ ID NO: 6), wherein thelinker portion of the fusion polypeptide comprises no more than 5, 4, 3,2 or 1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation). In someembodiments, the fusion polypeptide comprises an amino acid sequencethat is at least 85%, 90%, 95%, 97%, or 99% identical to any of theheterologous portion sequences disclosed herein (e.g., SEQ ID NO: 20),wherein the heterologous portion of the fusion polypeptide comprises nomore than 25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (butwhich may include further post-translational modifications, such asPEGylation). In some embodiments, the fusion polypeptide comprises anyof the TβRII polypeptide amino acid sequences disclosed herein (e.g.,SEQ ID NO: 18), wherein the TβRII polypeptide portion of the fusionpolypeptide comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation). In some embodiments, the fusionpolypeptide comprises any of the linker sequences disclosed herein(e.g., SEQ ID NO: 6), wherein the linker portion of the fusionpolypeptide comprises no more than 5, 4, 3, 2 or 1 additional aminoacids (but which may include further post-translational modifications,such as PEGylation). In some embodiments, the fusion polypeptidecomprises any of the heterologous portion sequences disclosed herein(e.g., SEQ ID NO: 20), wherein the heterologous portion of the fusionpolypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or 1additional amino acids (but which may include further post-translationalmodifications, such as PEGylation).

In some embodiments, the disclosure provides for a fusion polypeptide,wherein the fusion polypeptide consists or consists essentially of (andnot necessarily in the following order): a) an amino acid sequence thatis at least 85%, 90%, 95%, 97%, or 99% identical to any of the TβRIIpolypeptide amino acid sequences disclosed herein (e.g., SEQ ID NO: 18),wherein the TβRII polypeptide portion of the fusion polypeptidecomprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional aminoacids (but which may include further post-translational modifications,such as PEGylation); b) an amino acid sequence that is at least 85%,90%, 95%, 97%, or 99% identical to any of the linker sequences disclosedherein (e.g., SEQ ID NO: 6), wherein the linker portion of the fusionpolypeptide comprises no more than 5, 4, 3, 2 or 1 additional aminoacids (but which may include further post-translational modifications,such as PEGylation); and c) an amino acid sequence that is at least 85%,90%, 95%, 97%, or 99% identical to any of the heterologous portionsequences disclosed herein (e.g., SEQ ID NO: 20), wherein theheterologous portion of the fusion polypeptide comprises no more than25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which mayinclude further post-translational modifications, such as PEGylation);and d) optionally a leader sequence (e.g., SEQ ID NO: 23). In someembodiments, the disclosure provides for a fusion polypeptide, whereinthe fusion polypeptide consists or consists essentially of (and notnecessarily in the following order): a) any of the TβRII polypeptideamino acid sequences disclosed herein (e.g., SEQ ID NO: 18), wherein theTβRII polypeptide portion of the fusion polypeptide comprises no morethan 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional amino acids (but whichmay include further post-translational modifications, such asPEGylation); b) any of the linker sequences disclosed herein (e.g., SEQID NO: 6), wherein the linker portion of the fusion polypeptidecomprises no more than 5, 4, 3, 2 or 1 additional amino acids (but whichmay include further post-translational modifications, such asPEGylation); and c) any of the heterologous portion sequences disclosedherein (e.g., SEQ ID NO: 20), wherein the heterologous portion of thefusion polypeptide comprises no more than 25, 20, 15, 10, 5, 4, 3, 2, or1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation); and d) optionallya leader sequence (e.g., SEQ ID NO: 23).

In some embodiments, the disclosure provides for a fusion polypeptideconsisting of or consisting essentially of (and not necessarily in thefollowing order): a) a TβRII polypeptide portion consisting of an aminoacid sequence that is at least 85%, 90%, 95%, 97%, or 99% identical tothe amino acid sequence of SEQ ID NO: 18 and no more than 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation); b) a linkerportion consisting of an amino acid sequence that is at least 85%, 90%,95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 6and no more than 5, 4, 3, 2 or 1 additional amino acids (but which mayinclude further post-translational modifications, such as PEGylation);and c) a heterologous portion consisting of an amino acid sequence thatis at least 85%, 90%, 95%, 97%, or 99% identical to the amino acidsequence of SEQ ID NO: 20 and no more than 25, 20, 15, 10, 5, 4, 3, 2,or 1 additional amino acids (but which may include furtherpost-translational modifications, such as PEGylation); and d) optionallya leader sequence (e.g., SEQ ID NO: 23). In some embodiments, thedisclosure provides for a fusion polypeptide consisting or consistingessentially of (and not necessarily in the following order): a) a TβRIIpolypeptide portion consisting of the amino acid sequence of SEQ ID NO:18 and no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional aminoacids (but which may include further post-translational modifications,such as PEGylation); b) a linker portion consisting of the amino acidsequence of SEQ ID NO: 6 and no more than 5, 4, 3, 2 or 1 additionalamino acids (but which may include further post-translationalmodifications, such as PEGylation); and c) a heterologous portionconsisting of the amino acid sequence of SEQ ID NO: 20 and no more than25, 20, 15, 10, 5, 4, 3, 2, or 1 additional amino acids (but which mayinclude further post-translational modifications, such as PEGylation);and d) optionally a leader sequence (e.g., SEQ ID NO: 23).

In some embodiments, the fusion protein does not comprise a leadersequence. In some embodiments, the fusion protein comprises an aminoacid sequence that is at least 85%, 90%, 92%, 95%, 96%, 97%, 99%, or100% identical to the amino acid sequence of SEQ ID NO: 48.

(SEQ ID NO: 48) TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDTGGGGSGGGGSGGGGSGGGGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK.

In some embodiments, the disclosure provides for a TβRII fusionpolypeptide wherein the polypeptide does not comprise an antibody orantigen-binding portion thereof. In some embodiments, the polypeptidedoes not bind with appreciable affinity to a cytokine other than atransforming growth factor beta superfamily ligand (e.g., TGFβ1, TGFβ2and/or TGFβ3). In some embodiments, the polypeptide does not bind withappreciable affinity to a cytokine other than TGFβ1, TGFβ2 and/or TGFβ3.In some embodiments, the polypeptide does not bind with appreciableaffinity to a cytokine other than TGFβ1 and/or TGFβ3. In someembodiments, the polypeptide does not bind with appreciable affinity toCD4, CD8, CD25, CTLA-4, IL-10, TGFβ Receptor, PD-1, PD-L1, PD-L2, RANK,RANKL, HER2/neu, EGFR1, CD20, VEGF, TNF-α, TNFR2, FoxP3, CD80, CD86,IFN-α, IFN-β, IFN-γ, GITR, 4-1BB, OX-40, TLR1-10, ErbB-1, HER1,ErbB-3/HER3, ErbB-4/HER4, IGFR, IGFBP, IGF-1R, PDGFR, FGFR, VEGFR, HGFR,TRK receptor, ephrin receptors, AXL receptors, LTK receptors, TIEreceptors, angiopoietinl, 2, ROR receptor, DDR receptor, RET receptor,KLG receptor, RYK receptor, MuSK receptor, ILβR, IlαR, TNTRSF, TRAILreceptor, ARTC1, alpha-actinin-4, Bcr-abl, B-RAF, caspases,beta-catenin, fibronectin, GPNMB, GDP-L, LDLR, HLA-A2, MLA-A11, HSP70,KIAA205, MART2, MUM-1, 2, 3, PAP, neo-PAP, NFYC, OGT, OS-9, pml-RARalphafusion protein, PRDXS, PTPRK, KRAS2, NRAS, HRAS, RBAF600, SIRT2. SNRPD1,SYT-SSX1 or -SSX2 fusion protein, Triosephosphate Isomerase, BAGE,BAGE-1. BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V, HERV-K MEL,KK-LC, LAGE, LAGE-1, CAMEL, MAGE-1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS,MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-A11, MAGE-A12, MAGE-3,MAGE-B1, MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1),MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (S1LV), tyrosinase (TYR),TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17. SSX-1, 2, 3,4, TRP2-1NT2, carcino-embryonic antigen (CEA), Kallikfein 4,mammaglobm-A, OA1, prostate specific antigen (PSA), prostate specificmembrane antigen, TRP-1/, 75. TRP-2, AIM-2. BING-4, CPSF, cyclin D1,Ep-CAM, EpbA3, FGF-5, gp250, iCE), AFP, M-CSF, mdm-2, MUC1, p53 (TP53),PBF, FRAME, PSMA, RAGE-1. RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS),hTERT, telomerase, WT1, SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1,CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC,MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE,TPTE, estrogen receptors (ER), androgen receptors (AR), CD40, CD30,CD20, CD19, CD33, CD4, CD25, CD3, CA 72-4, CA 15-3, CA 27-29, CA 125, CA19-9, beta-human chorionic gonadotropin, 1-2 microglobulin, squamouscell carcinoma antigen, neuron-specific enoJase, heat shock proteingp96, GM2, sargramostim, CTLA-4, 707-AP, ART-4, CAP-1, CLCA2, Cyp-B,HST-2, HPV proteins, EBV proteins, Hepatitis B or C virus proteins,and/or HIV proteins.

In some embodiments, the disclosure provides for a TβRII fusionpolypeptide wherein the polypeptide does not comprise an additionalligand binding domain in addition to the TβRII domain. In someembodiments, the polypeptide comprises a linear amino acid sequencecomprising a TβRII domain and a heterologous portion (e.g., an Fcportion), but the linear amino acid sequence does not comprise anyadditional ligand binding domains. In some embodiments, the polypeptidecomprises a linear amino acid sequence comprising a TβRII domain and anFc portion, but the linear amino acid sequence does not comprise anyadditional ligand binding domains. In some embodiments, the disclosureprovides for a TβRII fusion polypeptide wherein the polypeptide does notcomprise multiple ligand binding domains in a single linear amino acidsequence. In some embodiments, the disclosure provides for a TβRIIfusion polypeptide wherein the polypeptide does not comprise more thanone continuous linker sequence in a single linear amino acid sequence.In some embodiments, the polypeptide does not comprise multiplecontinuous glycine and/or serine linkers (e.g., a linker comprising(GGGGS)n, wherein n=>4) (SEQ ID NO: 59)) in a single linear amino acidsequence. In some embodiments, the disclosure provides for a TβRIIfusion polypeptide wherein the heterologous portion is an Fc domain, andwherein only one continuous linker is covalently bound to the Fc domain.In some embodiments, the only one continuous linker comprises orconsists of a (GGGGS)n linker, wherein n=>4 (SEQ ID NO: 59).

6. Nucleic Acids and Methods of Manufacture

In certain embodiments, the present disclosure makes available isolatedand/or purified forms of the TβRII polypeptides fusion proteins, whichare isolated from, or otherwise substantially free of (e.g., at least80%, 90%, 95%, 96%, 97%, 98%, or 99% free of), other proteins and/orother TβRII polypeptide species. TβRII polypeptides will generally beproduced by expression from recombinant nucleic acids.

In certain embodiments, the disclosure includes nucleic acids encodingsoluble TβRII polypeptides comprising the coding sequence for anextracellular portion of a TβRII protein. In further embodiments, thisdisclosure also pertains to a host cell comprising such nucleic acids.The host cell may be any prokaryotic or eukaryotic cell. For example, apolypeptide of the present disclosure may be expressed in bacterialcells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art. Accordingly, some embodiments ofthe present disclosure further pertain to methods of producing the TβRIIpolypeptides.

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the TβRII polypeptides, includingfragments, functional variants and fusion proteins disclosed herein. SEQID NOs: 10, 12 and 14 encode variants of TβRII extracellular domainfused to an IgG Fc domain. The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids may be used, for example, in methods formaking TβRII polypeptides or as direct therapeutic agents (e.g., in anantisense, RNAi or gene therapy approach).

In certain aspects, the subject nucleic acids encoding TβRIIpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NOs: 10, 12 and 14. Variant nucleotide sequencesinclude sequences that differ by one or more nucleotide substitutions,additions or deletions, such as allelic variants.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NOs: 10, 12 and 14. In particularembodiments, the disclosure provides isolated or recombinant nucleicacid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 12, or fragments thereof. One ofordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NOs: 10, 12 and 14, and variants of SEQ ID NOs:10, 12 and 14 are also within the scope of this disclosure. In furtherembodiments, the nucleic acid sequences of the disclosure can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the disclosure also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequences designated in SEQ ID NOs: 10, 12 and 14complement sequences of SEQ ID NOs: 10, 12 and 14, or fragments thereof.As discussed above, one of ordinary skill in the art will understandreadily that appropriate stringency conditions which promote DNAhybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In some embodiments, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 10, 12 and 14 due to degeneracy in the genetic code arealso within the scope of the disclosure. For example, a number of aminoacids are designated by more than one triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this disclosure.

It will be appreciated by one of skill in the art that correspondingvariants based on the long isoform of TβRII will include nucleotidesequences encoding the 25-amino acid insertion along with a conservativeVal-Ile substitution at the flanking position C-terminal to theinsertion. It will also be appreciated that corresponding variants basedon either the long (A) or short (B) isoforms of TβRII will includevariant nucleotide sequences comprising an insertion of 108 nucleotides,encoding a 36-amino-acid insertion (SEQ ID NO: 41), at the same locationdescribed for naturally occurring TβRII isoform C.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects disclosed herein, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a TβRII polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the TβRII polypeptide. Accordingly, theterm regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding a TβRII polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid included in the disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant TβRII polypeptide include plasmids and other vectors.For instance, suitable vectors include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the B-gal containing pBlueBac III).

In certain embodiments, a vector will be designed for production of thesubject TβRII polypeptides in CHO cells, such as a Pcmv-Script vector(Stratagene, La Jolla, Calif.), pcDN4 vectors (Invitrogen, Carlsbad,Calif.) and pCI-neo vectors (Promega, Madison, Wis.). In a preferredembodiment, a vector will be designed for production of the subjectTβRII polypeptides in HEK-293 cells. As will be apparent, the subjectgene constructs can be used to cause expression of the subject TβRIIpolypeptides in cells propagated in culture, e.g., to produce proteins,including fusion proteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NOs: 10, 12,or 14) for one or more of the subject TβRII polypeptides. The host cellmay be any prokaryotic or eukaryotic cell. For example, a TβRIIpolypeptide disclosed herein may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject TβRII polypeptides. For example, a host celltransfected with an expression vector encoding a TβRII polypeptide canbe cultured under appropriate conditions to allow expression of theTβRII polypeptide to occur. The TβRII polypeptide may be secreted andisolated from a mixture of cells and medium containing the TβRIIpolypeptide. Alternatively, the TβRII polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, and media.Suitable media for cell culture are well known in the art. The subjectTβRII polypeptides can be isolated from cell culture medium, host cells,or both, using techniques known in the art for purifying proteins,including ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, immunoaffinity purification withantibodies specific for particular epitopes of the TβRII polypeptidesand affinity purification with an agent that binds to a domain fused tothe TβRII polypeptide (e.g., a protein A column may be used to purify anTβRII-Fc fusion). In a preferred embodiment, the TβRII polypeptide is afusion protein containing a domain which facilitates its purification.As an example, purification may be achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant TβRIIpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified TβRII polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

7. Alterations in Fe-Fusion Proteins

The application further provides TβRII-Fc fusion proteins withengineered or variant Fc regions. Such antibodies and Fc fusion proteinsmay be useful, for example, in modulating effector functions, such as,antigen-dependent cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). Additionally, the modifications may improve thestability of the antibodies and Fc fusion proteins. Amino acid sequencevariants of the antibodies and Fc fusion proteins are prepared byintroducing appropriate nucleotide changes into the DNA, or by peptidesynthesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibodies and Fc fusion proteins disclosed herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibodies and Fc fusion proteins,such as changing the number or position of glycosylation sites.

Antibodies and Fc fusion proteins with reduced effector function may beproduced by introducing changes in the amino acid sequence, including,but are not limited to, the Ala-Ala mutation described by Bluestone etal. (see WO 94/28027 and WO 98/47531; also see Xu et al. 2000 CellImmunol 200; 16-26). Thus, in certain embodiments, Fc fusion proteins ofthe disclosure with mutations within the constant region including theAla-Ala mutation may be used to reduce or abolish effector function.According to these embodiments, antibodies and Fc fusion proteins maycomprise a mutation to an alanine at position 234 or a mutation to analanine at position 235, or a combination thereof. In one embodiment,the antibody or Fc fusion protein comprises an IgG4 framework, whereinthe Ala-Ala mutation would describe a mutation(s) from phenylalanine toalanine at position 234 and/or a mutation from leucine to alanine atposition 235. In another embodiment, the antibody or Fc fusion proteincomprises an IgG1 framework, wherein the Ala-Ala mutation would describea mutation(s) from leucine to alanine at position 234 and/or a mutationfrom leucine to alanine at position 235. The antibody or Fc fusionprotein may alternatively or additionally carry other mutations,including the point mutation K322A in the CH2 domain (Hezareh et al.2001 J Virol. 75: 12161-8).

In particular embodiments, the antibody or Fc fusion protein may bemodified to either enhance or inhibit complement dependent cytotoxicity(CDC). Modulated CDC activity may be achieved by introducing one or moreamino acid substitutions, insertions, or deletions in an Fc region (see,e.g., U.S. Pat. No. 6,194,551). Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved or reduced internalizationcapability and/or increased or decreased complement-mediated cellkilling. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes,B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature322: 738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351.

8. Screening Assays

In certain aspects, the present invention relates to the use of TβRIIpolypeptides (e.g., soluble TβRII polypeptides) to identify compounds(agents) which are agonist or antagonists of the TGFβ1, TGFβ3 and TβRIIsignaling pathway. Compounds identified through this screening can betested to assess their ability to modulate TGFβ1 and TGFβ3 signalingactivity in vitro. Optionally, these compounds can further be tested inanimal models to assess their ability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting TGFβ1, TGFβ3 and TβRIIpolypeptides. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturb TGFβ1,TGFβ3 or TβRII-mediated cell signaling. In certain embodiments, theassay is carried out to screen and identify compounds that specificallyinhibit or reduce binding of a TβRII polypeptide to TGFβ1 or TGFβ3.Alternatively, the assay can be used to identify compounds that enhancebinding of a TβRII polypeptide to TGFβ1 or TGFβ3. In a furtherembodiment, the compounds can be identified by their ability to interactwith a TGFβ1, TGFβ3 or TβRII polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a TβRIIpolypeptide and TGFβ1 or TGFβ3.

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified TβRII polypeptide which is ordinarily capable of binding toTGFβ1 or TGFβ3. To the mixture of the compound and TβRII polypeptide isthen added a composition containing a TβRII ligand. Detection andquantification of TβRII/TGFβ1 or TβRII/TGFβ3 complexes provides a meansfor determining the compound's efficacy at inhibiting (or potentiating)complex formation between the TβRII polypeptide and TGFβ1 or TGFβ3. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. For example, in a control assay, isolated and apurified TGFβ1 or TGFβ3 is added to a composition containing the TβRIIpolypeptide, and the formation of TβRII/TGFβ1 or TβRII/TGFβ3 complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the TβRII polypeptide and TGFβ1 or TGFβ3 maybe detected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled TβRIIpolypeptide or TGFβ1 or TGFβ3, by immunoassay, or by chromatographicdetection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a TβRII polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a TβRII polypeptide andits binding protein. See for example, U.S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between a TβRII polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with a TβRII or TGFβ1 or TGFβ3 polypeptide of theinvention. The interaction between the compound and the TβRII or TGFβ1or TGFβ3 polypeptide may be covalent or non-covalent. For example, suchinteraction can be identified at the protein level using in vitrobiochemical methods, including photo-crosslinking, radiolabeled ligandbinding, and affinity chromatography (Jakoby W B et al., 1974, Methodsin Enzymology 46: 1). In certain cases, the compounds may be screened ina mechanism based assay, such as an assay to detect compounds which bindto a TGFβ1 or TGFβ3 or TβRII polypeptide. This may include a solid-phaseor fluid-phase binding event. Alternatively, the gene encoding a TGFβ1or TGFβ3 or TβRII polypeptide can be transfected with a reporter system(e.g., β-galactosidase, luciferase, or green fluorescent protein) into acell and screened against the library preferably by a high-throughputscreening or with individual members of the library. Othermechanism-based binding assays may be used, for example, binding assayswhich detect changes in free energy. Binding assays can be performedwith the target fixed to a well, bead or chip or captured by animmobilized antibody or resolved by capillary electrophoresis. The boundcompounds may be detected usually using colorimetric or fluorescence orsurface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) TGFβ1- or TGFβ3-mediated cellsignaling. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate TGFβ1 or TGFβ3 signaling. Various methods known in the art canbe utilized for this purpose.

9. Exemplary Therapeutic Uses

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The terms “treatment”, “treating”, “alleviation” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect, and may also be used to refer to improving,alleviating, and/or decreasing the severity of one or more symptoms of acondition being treated. The effect may be prophylactic in terms ofcompletely or partially delaying the onset or recurrence of a disease,condition, or symptoms thereof, and/or may be therapeutic in terms of apartial or complete cure for a disease or condition and/or adverseeffect attributable to the disease or condition. “Treatment” as usedherein covers any treatment of a disease or condition of a mammal,particularly a human, and includes: (a) preventing the disease orcondition from occurring in a subject which may be predisposed to thedisease or condition but has not yet been diagnosed as having it; (b)inhibiting the disease or condition (e.g., arresting its development);or (c) relieving the disease or condition (e.g., causing regression ofthe disease or condition, providing improvement in one or moresymptoms).

The terms “patient”, “subject”, or “individual” are used interchangeablyherein and refer to either a human or a non-human animal. These termsinclude mammals, such as humans, non-human primates, laboratory animals,livestock animals (including bovines, porcines, camels, etc.), companionanimals (e.g., canines, felines, other domesticated animals, etc.) androdents (e.g., mice and rats). In particular embodiments, the patient,subject or individual is a human.

The disclosure provides methods of treating or preventing a disease orcondition associated with a TGFβ superfamily member by administering toa subject an effective amount of a TβRII polypeptide, including aTβRII-Fc fusion protein of the foregoing, hereafter collectivelyreferred to as “therapeutic agents”. In some embodiments the disease orcondition is associated with dysregulated TGFβ1 or TGFβ3 signaling. Alsoprovided are methods and compositions for treating certaincardiovascular or vascular disorders. In addition, the disclosureprovides methods and compositions for treating or preventing cancer. Inaddition, the disclosure provides methods and compositions for treatingor preventing fibrotic disorders and conditions.

In particular, polypeptide therapeutic agents of the present disclosureare useful for treating or preventing chronic vascular or cardiovasculardiseases. Exemplary disorders of this kind include, but are not limitedto, heart disease (including myocardial disease, myocardial infarct,angina pectoris, and heart valve disease); renal disease (includingchronic glomerular inflammation, diabetic renal failure, andlupus-related renal inflammation); disorders associated withatherosclerosis or other types of arteriosclerosis (including stroke,cerebral hemorrhage, subarachnoid hemorrhage, angina pectoris, and renalarteriosclerosis); thrombotic disorders (including cerebral thrombosis,thrombotic intestinal necrosis); complications of diabetes (includingdiabetes-related retinal disease, cataracts, diabetes-related renaldisease, diabetes-related neuropathology, diabetes-related gangrene, anddiabetes-related chronic infection); vascular inflammatory disorders(systemic lupus erythematosus, joint rheumatism, joint arterialinflammation, large-cell arterial inflammation, Kawasaki disease,Takayasu arteritis, Churg-Strauss syndrome, and Henoch-Schoenleinpurpura); diabetic vasculopathies; and cardiac disorders such ascongenital heart disease, cardiomyopathy (e.g., dilated, hypertrophic,restrictive cardiomyopathy), and congestive heart failure. Exemplarydisorders further include, but are not limited to, hereditaryhemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome,familial thoracic aortic aneurysm syndrome, arterial tortuositysyndrome, pre-eclampsia, and restenosis.

The TβRII polypeptide can be administered to the subject alone, or incombination with one or more agents or therapeutic modalities, e.g.,therapeutic agents, which are useful for treating TGFβ associatedcardiovascular disorders and/or conditions. In certain embodiments, thesecond agent or therapeutic modality is chosen from one or more of:angioplasty, beta blockers, anti-hypertensives, cardiotonics,anti-thrombotics, vasodilators, hormone antagonists, endothelinantagonists, calcium channel blockers, phosphodiesterase inhibitors,angiotensin type 2 antagonists and/or cytokine blockers/inhibitors

In particular, polypeptide therapeutic agents of the present disclosureare useful for treating or preventing a cancer (tumor). The terms“cancer” and “cancerous” refer to or describe, the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth/proliferation. Examples of cancer, or neoplastic disorders,include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, stomach cancer,intestinal cancer, skin cancer, bone cancer, gastric cancer, melanoma,and various types of head and neck cancer, including squamous cell headand neck cancer. Other examples of neoplastic disorders and relatedconditions include esophageal carcinomas, thecomas, arrhenoblastomas,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, Wilm'stumor, renal cell carcinoma, prostate carcinoma, abnormal vascularproliferation associated with phakomatoses, and Meigs' syndrome. Acancer that is particularly amenable to treatment with the therapeuticagents described herein may be characterized by one or more of thefollowing: the cancer has elevated TβRII levels detectable in the tumoror the serum, increased TGFβ1 or TGFβ3 expression levels or biologicalactivity, is metastatic or at risk of becoming metastatic, or anycombination thereof.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject methods of the disclosure can beused alone. Alternatively, the subject methods may be used incombination with other conventional anti-cancer therapeutic approachesdirected to treatment or prevention of proliferative disorders (e.g.,tumor). For example, such methods can be used in prophylactic cancerprevention, prevention of cancer recurrence and metastases aftersurgery, and as an adjuvant of other conventional cancer therapy. Thepresent disclosure recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery) can be enhanced through the use of a subjectpolypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic or anti-cancer activities. These compounds have beenused as pharmaceutical agents in chemotherapy to shrink solid tumors,prevent metastases and further growth, or decrease the number ofmalignant cells in leukemic or bone marrow malignancies. Althoughchemotherapy has been effective in treating various types ofmalignancies, many anti-neoplastic compounds induce undesirable sideeffects. It has been shown that when two or more different treatmentsare combined, the treatments may work synergistically and allowreduction of dosage of each of the treatments, thereby reducing thedetrimental side effects exerted by each compound at higher dosages. Inother instances, malignancies that are refractory to a treatment mayrespond to a combination therapy of two or more different treatments.

When a therapeutic agent disclosed herein is administered in combinationwith another conventional anti-neoplastic agent, either concomitantly orsequentially, such therapeutic agent may enhance the therapeutic effectof the anti-neoplastic agent or overcome cellular resistance to suchanti-neoplastic agent. This allows decrease of dosage of ananti-neoplastic agent, thereby reducing the undesirable side effects, orrestores the effectiveness of an anti-neoplastic agent in resistantcells.

According to the present disclosure, the polypeptide therapeutic agentsdescribed herein may be used in combination with other compositions andprocedures for the treatment of diseases. For example, a tumor may betreated conventionally with surgery, radiation or chemotherapy combinedwith the TβRII polypeptide, and then the TβRII polypeptide may besubsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize any residual primary tumor.

In certain aspects of the invention, other therapeutic agents useful forcombination tumor therapy with a TβRII polypeptide include other cancertherapies: e.g., surgery, cytotoxic agents, radiological treatmentsinvolving irradiation or administration of radioactive substances,chemotherapeutic agents, anti-hormonal agents, growth inhibitory agents,anti-neoplastic compositions, and treatment with anti-cancer agentslisted herein and known in the art, or combinations thereof.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cyclophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as folinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elfornithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylomithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY1 17018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In addition,such definition of chemotherapeutic agents includes bisphosphonates suchas clodronate (for example, BONEFOS® or OSTAC®), DIDROC AL® etidronate,NE-58095, ZOMET zoledronic acid/zoledronate, FOSAMAX® alendronate,AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);antisense oligonucleotides, particularly those that inhibit expressionof genes in signaling pathways implicated in aberrant cellproliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as THERATOPE®vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine,LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1inhibitor; ABARELIX® rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dualtyrosine kinase small-molecule inhibitor also known as GW572016); andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

In still other embodiments, TβRII polypeptides may be useful in thetreatment or prevention of fibrosis. As used herein, the term “fibrosis”refers to the aberrant formation or development of excess fibrousconnective tissue by cells in an organ or tissue. Although processesrelated to fibrosis can occur as part of normal tissue formation orrepair, dysregulation of these processes can lead to altered cellularcomposition and excess connective tissue deposition that progressivelyimpairs to tissue or organ function. The formation of fibrous tissue canresult from a reparative or reactive process. Fibrotic disorders orconditions include, but are not limited to, fibroproliferative disordersassociated with vascular diseases, such as cardiac disease, cerebraldisease, and peripheral vascular disease, as well as tissues and organsystems including the heart, skin, kidney, peritoneum, gut, and liver(as disclosed in, e.g., Wynn, 2004, Nat Rev 4:583-594, incorporatedherein by reference). Exemplary disorders that can be treated include,but are not limited to, renal fibrosis, including nephropathiesassociated with injury/fibrosis, e.g., chronic nephropathies associatedwith diabetes (e.g., diabetic nephropathy), lupus, scleroderma,glomerular nephritis, focal segmental glomerular sclerosis, and IgAnephropathy; gut fibrosis, e.g., scleroderma, and radiation-induced gutfibrosis; liver fibrosis, e.g., cirrhosis, alcohol-induced liverfibrosis, biliary duct injury, primary biliary cirrhosis, infection orviral-induced liver fibrosis, congenital hepatic fibrosis and autoimmunehepatitis; and other fibrotic conditions, such as cystic fibrosis,endomyocardial fibrosis, mediastinal fibrosis, sarcoidosis, scleroderma,spinal cord injury/fibrosis, myelofibrosis, vascular restenosis,atherosclerosis, injection fibrosis (which can occur as a complicationof intramuscular injections, especially in children), endomyocardialfibrosis, retroperitoneal fibrosis, and nephrogenic systemic fibrosis.

As used herein, the terms “fibrotic disorder”, “fibrotic condition,” and“fibrotic disease,” are used interchangeably to refer to a disorder,condition or disease characterized by fibrosis. Examples of fibroticdisorders include, but are not limited to lupus, sclerotic disorders(e.g., scleroderma, atherosclerosis, and systemic scleroisis including,e.g., diffuse systemic sclerosis and progressive systemic sclerosis),vascular fibrosis, pancreatic fibrosis, liver fibrosis (e.g.,cirrhosis), renal fibrosis, musculoskeletal fibrosis, cardiac fibrosis(e.g., endomyocardial fibrosis, idiopathic myocardiopathy), skinfibrosis (e.g., scleroderma, post-traumatic, operative cutaneousscarring, keloids and cutaneous keloid formation), eye fibrosis (e.g.,glaucoma, sclerosis of the eyes, conjunctival and corneal scarring, andpterygium), myelofibrosis, chronic graft-versus-host disease, Peyronie'sdisease, post-cystoscopic urethral stenosis, idiopathic andpharmacologically induced retroperitoneal fibrosis, mediastinalfibrosis, proliferative fibrosis, neoplastic fibrosis, Dupuytren'sdisease, strictures, neural scarring, dermal scarring, idiopathicpulmonary fibrosis and radiation induced fibrosis.

In some embodiments, any of the polypeptides disclosed herein (e.g., apolypeptide having the amino acid sequence of any one of SEQ ID NOs: 13and 50-56) may be used, alone or in combination with one or moresupportive therapies or active agents, to treat, prevent, or reduce theprogression rate and/or severity of an interstitial lung disease (e.g.,idiopathic pulmonary fibrosis). In some embodiments, the interstitiallung disease is pulmonary fibrosis. In some embodiments, theinterstitial lung disease is caused by any one of the following:silicosis, asbestosis, berylliosis, hypersensitivity pneumonitis, druguse (e.g., antibiotics, chemotherapeutic drugs, antiarrhythmic agents,statins), systemic sclerosis, polymyositis, dermatomyositis, systemiclupus erythematosus, rheumatoid arthritis, an infection (e.g., atypicalpneumonia, pneumocystis pneumonia, tuberculosis, Chlamydia trachomatis,and/or respiratory syncytial virus), lymphangitic carcinomatosis,cigarette smoking, or developmental disorders. In some embodiments, theinterstitial lung disease is idiopathic (e.g., sarcoidosis, idiopathicpulmonary fibrosis, Hamman-Rich syndrome, and/or antisynthetasesyndrome). In particular embodiments, the interstitial lung disease isidiopathic pulmonary fibrosis. In some embodiments, the treatment foridiopathic pulmonary fibrosis is administered in combination with anadditional therapeutic agent. In some embodiments, the additionaltherapeutic agent is selected from the group consisting of: pirfenidone,N-acetylcysteine, prednisone, azathioprine, nintedanib, derivativesthereof and combinations thereof.

In some embodiments, any of the polypeptides disclosed herein (e.g., apolypeptide having the amino acid sequence of any one of SEQ ID NOs: 13and 50-56) may be used, alone or in combination with one or moresupportive therapies or active agents, to treat, prevent, or reduce theprogression rate and/or severity of a kidney-associated disease orcondition. As used herein, “kidney-associated disease or condition” canrefer to any disease, disorder, or condition that affects the kidneys orthe renal system. Examples of kidney-associated diseases or conditionsinclude, but are not limited to, chronic kidney diseases (or failure),acute kidney diseases (or failure), primary kidney diseases,non-diabetic kidney diseases, glomerulonephritis, interstitialnephritis, diabetic kidney diseases, diabetic nephropathy,glomerulosclerosis, rapid progressive glomerulonephritis, renalfibrosis, Alport syndrome, IDDM nephritis, mesangial proliferativeglomerulonephritis, membranoproliferative glomerulonephritis, crescenticglomerulonephritis, renal interstitial fibrosis, focal segmentalglomerulosclerosis, membranous nephropathy, minimal change disease,pauci-immune rapid progressive glomerulonephritis, IgA nephropathy,polycystic kidney disease, Dent's disease, nephrocytinosis, Heymannnephritis, autosomal dominant (adult) polycystic kidney disease,autosomal recessive (childhood) polycystic kidney disease, acute kidneyinjury, nephrotic syndrome, renal ischemia, podocyte diseases ordisorders, proteinuria, glomerular diseases, membranousglomerulonephritis, focal segmental glomerulonephritis, pre-eclampsia,eclampsia, kidney lesions, collagen vascular diseases, benignorthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, acute interstitial nephritis, establishedchronic kidney disease, renal artery stenosis, ischemic nephropathy,uremia, drug and toxin-induced chronic tubulointerstitial nephritis,reflux nephropathy, kidney stones, Goodpasture's syndrome, normocyticnormochromic anemia, renal anemia, diabetic chronic kidney disease,IgG4-related disease, von Hippel-Lindau syndrome, tuberous sclerosis,nephronophthisis, medullary cystic kidney disease, renal cell carcinoma,adenocarcinoma, nephroblastoma, lymphoma, leukemia, hyposialylationdisorder, chronic cyclosporine nephropathy, renal reperfusion injury,renal dysplasia, azotemia, bilateral arterial occlusion, acute uric acidnephropathy, hypovolemia, acute bilateral obstructive uropathy,hypercalcemic nephropathy, hemolytic uremic syndrome, acute urinaryretention, malignant nephrosclerosis, postpartum glomerulosclerosis,scleroderma, non-Goodpasture's anti-GBM disease, microscopicpolyarteritis nodosa, allergic granulomatosis, acute radiationnephritis, post-streptococcal glomerulonephritis, Waldenstrom'smacroglobulinemia, analgesic nephropathy, arteriovenous fistula,arteriovenous graft, dialysis, ectopic kidney, medullary sponge kidney,renal osteodystrophy, solitary kidney, hydronephrosis, microalbuminuria,uremia, haematuria, hyperlipidemia, hypoalbuminaemia, lipiduria,acidosis, hyperkalemia, and edema.

In some embodiments, any of the polypeptides disclosed herein (e.g., apolypeptide having the amino acid sequence of any one of SEQ ID NOs: 13and 50-56) may be used, alone or in combination with one or moresupportive therapies or active agents, to treat, prevent, or reduce theprogression rate and/or severity of chronic kidney disease (e.g., tissuedamage, inflammation, and/or fibrosis). Chronic kidney disease (CKD),also known as chronic renal disease, is a progressive loss in renalfunction over a period of months or years. The symptoms of worseningkidney function may include feeling generally unwell and experiencing areduced appetite. Often, chronic kidney disease is diagnosed as a resultof screening of people known to be at risk of kidney problems, such asthose with high blood pressure or diabetes and those with a bloodrelative with CKD. This disease may also be identified when it leads toone of its recognized complications, such as cardiovascular disease,anemia, or pericarditis. Recent professional guidelines classify theseverity of CKD in five stages, with stage 1 being the mildest andusually causing few symptoms and stage 5 being a severe illness withpoor life expectancy if untreated. Stage 5 CKD is often called end-stagekidney disease, end-stage renal disease, or end-stage kidney failure,and is largely synonymous with the now outdated terms chronic renalfailure or chronic kidney failure; and usually means the patientrequires renal replacement therapy, which may involve a form ofdialysis, but ideally constitutes a kidney transplant. CKD is initiallywithout specific symptoms and is generally only detected as an increasein serum creatinine or protein in the urine. As the kidney functiondecreases, various symptoms may manifest as described below. Bloodpressure may be increased due to fluid overload and production ofvasoactive hormones created by the kidney via the renin-angiotensinsystem, increasing one's risk of developing hypertension and/orsuffering from congestive heart failure. Urea may accumulate, leading toazotemia and ultimately uremia (symptoms ranging from lethargy topericarditis and encephalopathy). Due to its high systemic circulation,urea is excreted in eccrine sweat at high concentrations andcrystallizes on skin as the sweat evaporates (“uremic frost”). Potassiummay accumulate in the blood (hyperkalemia with a range of symptomsincluding malaise and potentially fatal cardiac arrhythmias).Hyperkalemia usually does not develop until the glomerular filtrationrate falls to less than 20-25 ml/min/1.73 m2, at which point the kidneyshave decreased ability to excrete potassium. Hyperkalemia in CKD can beexacerbated by acidemia (which leads to extracellular shift ofpotassium) and from lack of insulin. Erythropoietin synthesis may bedecreased causing anemia. Fluid volume overload symptoms may occur,ranging from mild edema to life-threatening pulmonary edema.Hyperphosphatemia, due to reduced phosphate excretion, may occurgenerally following the decrease in glomerular filtration.Hyperphosphatemia is associated with increased cardiovascular risk,being a direct stimulus to vascular calcification. Hypocalcemia maymanifest, which is generally caused by stimulation of fibroblast growthfactor-23. Osteocytes are responsible for the increased production ofFGF23, which is a potent inhibitor of the enzyme 1-alpha-hydroxylase(responsible for the conversion of 25-hydroxycholecalciferol into 1,25dihydroxyvitamin D3). Later, this progresses to secondaryhyperparathyroidism, renal osteodystrophy, and vascular calcificationthat further impairs cardiac function. Metabolic acidosis (due toaccumulation of sulfates, phosphates, uric acid etc.) may occur andcause altered enzyme activity by excess acid acting on enzymes; and alsoincreased excitability of cardiac and neuronal membranes by thepromotion of hyperkalemia due to excess acid (acidemia). Acidosis isalso due to decreased capacity to generate enough ammonia from the cellsof the proximal tubule. Iron deficiency anemia, which increases inprevalence as kidney function decreases, is especially prevalent inthose requiring haemodialysis. It is multifactoral in cause, butincludes increased inflammation, reduction in erythropoietin, andhyperuricemia leading to bone marrow suppression. People with CKD sufferfrom accelerated atherosclerosis and are more likely to developcardiovascular disease than the general population. Patients afflictedwith CKD and cardiovascular disease tend to have significantly worseprognoses than those suffering only from the latter. In someembodiments, the chronic kidney disease is a chronic kidney diseasemineral bone disorder, a broad syndrome of interrelated skeletal,cardiovascular, and mineral-metabolic disorders arising from kidneydisease. CKD-MBD encompasses various skeletal pathologies often referredto as renal osteodystrophy (ROD), which is a preferred embodiment fortreatment with any of the polypeptides disclosed herein, or combinationswith one or more supportive therapies or active agents. Depending on therelative contribution of different pathogenic factors, ROD is manifestedas diverse pathologic patterns of bone remodeling (Hruska et al., 2008,Chronic kidney disease mineral bone disorder (CKD-MBD); in Rosen et al.(ed) Primer on the Metabolic Bone Diseases and Disorders of MineralMetabolism, 7th ed. American Society for Bone and Mineral Research,Washington D.C., pp 343-349). At one end of the spectrum is ROD withuremic osteodystrophy and low bone turnover, characterized by a lownumber of active remodeling sites, profoundly suppressed bone formation,and low bone resorption. At the other extreme is ROD withhyperparathyroidism, high bone turnover, and osteitis fibrosa.

In certain aspects, any of the polypeptides (e.g., a polypeptide havingthe amino acid sequence of SEQ ID NOs: 13 and 50-56) disclosed hereinmay be used, alone or in combination with one or more supportivetherapies or active agents, to treat, prevent, or reduce the progressionrate and/or severity of myelofibrosis (e.g., primary myelofibrosis,post-polycythemia vera myelofibrosis, and post-essential thrombocythemiamyelofibrosis). In particular, ActRIIB antagonists may be used, alone orin combination with one or more supportive therapies or active agents,to treat, prevent, or reduce the progression rate and/or severity of oneor more complications of myelofibrosis including, for example,ineffective hematopoiesis, anemia, inflammation, fibrosis (e.g., bonemarrow fibrosis, spleen fibrosis, and liver fibrosis), pancytopenia,thrombocytopenia, extramedullary hematopoiesis (e.g., splenicextramedullary hematopoiesis, hepatic extramedullary hematopoiesis,pulmonary extramedullary hematopoiesis, and lymphatic extramedullaryhematopoiesis), hepatomegaly, splenomegaly, osteosclerosis,osteomyelofibrosis, poikilocytosis, fatigue, weight loss, night sweats,fever, pruritus, bone pain, early satiety, abdominal pain or discomfort,arthralgias, myalgias, parasthesias, cachexia, splenic infarct, andbleeding.

As used herein, inhibition of the fibrotic response of a cell, includes,but is not limited to the inhibition of the fibrotic response of one ormore cells within the liver (or liver tissue); one or more cells withinthe kidney (or renal tissue); one or more cells within muscle tissue;one or more cells within the heart (or cardiac tissue); one or morecells within the pancreas; one or more cells within the skin; one ormore cells within the bone, one or more cells within the vasculature,one or more stem cells, or one or more cells within the eye.

In some embodiments, any of the TβRII polypeptides of the disclosure maybe used for treating an autoimmune disease or disorder. In someembodiments, the autoimmune disease or disorder is selected from thegroup consisting of: spondyloarthropathies; ankylosing spondylitis,arthritis, psoriatic arthritis/spondylitis, enteropathic arthritis,reactive arthritis, Reiter's syndrome, undifferentiatedspondyloarthropathies; reactive arthritis, rheumatism, inflammatorybowel syndrome, Crohns Disease, rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis, gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis and nephroticsyndrome.

In some embodiments, any of the TβRII polypeptides of the disclosure maybe used for treating a metabolic disorder. In some embodiments, themetabolic disorder is obesity or diabetes (e.g., Type I or Type IIdiabetes), fatty liver disease, diabetic neuropathy, peripheralneuropathy, diabetic retinopathy, diabetic ulcerations, retinopathyulcerations, diabetic macrovasculopathy.

In some embodiments, any of the TβRII polypeptides of the disclosure maybe used for treating an infectious disease or following an organ ortissue transplantation.

In some embodiments, any of the TβRII polypeptides of the disclosure maybe used for treating chronic obstructive pulmonary disease (COPD),chronic obstructive airway disorder, idiopathic pulmonary fibrosisand/or asthma. In part, the disclosure also relates to methods oftreating pulmonary hypertension (e.g., pulmonary arterial hypertension)comprising administering to a patient in need thereof an effectiveamount of a TβRII polypeptide (e.g., a polypeptide comprising the aminoacid sequence of any one of SEQ ID NOs: 13 and 50-56). In someembodiments, the disclosure contemplates methods of treating one or morecomplications of pulmonary hypertension (e.g., smooth muscle and/orendothelial cell proliferation in the pulmonary artery, angiogenesis inthe pulmonary artery, dyspnea, chest pain, pulmonary vascularremodeling, right ventricular hypertrophy, and pulmonary fibrosis)comprising administering to a patient in need thereof an effectiveamount of a TβRII polypeptide. In some embodiments, the disclosurecontemplates methods of preventing one or more complications ofpulmonary hypertension comprising administering to a patient in needthereof an effective amount of a TβRII polypeptide. In some embodiments,the disclosure contemplates methods of reducing the progression rate ofpulmonary hypertension comprising administering to a patient in needthereof an effective amount of a TβRII polypeptide. In some embodiments,the disclosure contemplates methods of reducing the progression rate ofone or more complications of pulmonary hypertension comprisingadministering to a patient in need thereof an effective amount of aTβRII polypeptide. In some embodiments, the disclosure contemplatesmethods of reducing the severity of pulmonary hypertension comprisingadministering to a patient in need thereof an effective amount of aTβRII polypeptide. In some embodiments, the disclosure contemplatesmethods of reducing the severity of one or more complications ofpulmonary hypertension comprising administering to a patient in needthereof an effective amount of a TβRII polypeptide. Optionally, methodsdisclosed herein for treating, preventing, or reducing the progressionrate and/or severity of pulmonary hypertension, particularly treating,preventing, or reducing the progression rate and/or severity of one ormore complications of pulmonary hypertension, may further compriseadministering to the patient one or more supportive therapies oradditional active agents for treating pulmonary hypertension.

The present invention contemplates the use of TβRII polypeptides incombination with one or more other therapeutic modalities. Thus, inaddition to the use of TβRII polypeptides, one may also administer tothe subject one or more “standard” therapies for treating fibroticdisorders. For example, the TβRII polypeptides can be administered incombination with (i.e., together with) cytotoxins, immunosuppressiveagents, radiotoxic agents, and/or therapeutic antibodies. Particularco-therapeutics contemplated by the present invention include, but arenot limited to, steroids (e.g., corticosteroids, such as Prednisone),immune-suppressing and/or anti-inflammatory agents (e.g.,gamma-interferon, cyclophosphamide, azathioprine, methotrexate,penicillamine, cyclosporine, colchicine, antithymocyte globulin,mycophenolate mofetil, and hydroxychloroquine), cytotoxic drugs, calciumchannel blockers (e.g., nifedipine), angiotensin converting enzymeinhibitors (ACE) inhibitors, para-aminobenzoic acid (PABA), dimethylsulfoxide, transforming growth factor beta (TGFβ) inhibitors,interleukin-5 (IL-5) inhibitors, and pan caspase inhibitors.

Additional anti-fibrotic agents that may be used in combination withTβRII polypeptides include, but are not limited to, lectins (asdescribed in, for example, U.S. Pat. No. 7,026,283, the entire contentsof which is incorporated herein by reference), as well as theanti-fibrotic agents described by Wynn et al (2007, J Clin Invest117:524-529, the entire contents of which is incorporated herein byreference). For example, additional anti-fibrotic agents and therapiesinclude, but are not limited to, variousanti-inflammatory/immunosuppressive/cytotoxic drugs (includingcolchicine, azathioprine, cyclophosphamide, prednisone, thalidomide,pentoxifylline and theophylline), TGFβ signaling modifiers (includingrelaxin, SMAD7, HGF, and BMP7, as well as TGFβ1, TβRII, EGR-I, and CTGFinhibitors), cytokine and cytokine receptor antagonists (inhibitors ofIL-1β, IL-5, IL-6, IL-13, IL-21, IL-4R, IL-13Rα1, GM-CSF, TNF-α,oncostatin M, W1SP-I, and PDGFs), cytokines and chemokincs (IFN-γ,IFN-α/β, IL-12, IL-10, HGF, CXCL10, and CXCL11), chemokine antagonists(inhibitors of CXCL1, CXCL2, CXCL12, CCL2, CCL3, CCL6, CCL17, andCCL18), chemokine receptor antagonists (inhibitors of CCR2, CCR3, CCRS,CCR7, CXCR2, and CXCR4), TLR antagonists (inhibitors of TLR3, TLR4, andTLR9), angiogenesis antagonists (VEGF-specific antibodies and adenosinedeaminase replacement therapy), antihypertensive drugs (beta blockersand inhibitors of ANG 11, ACE, and aldosterone), vasoactive substances(ET-1 receptor antagonists and bosetan), inhibitors of the enzymes thatsynthesize and process collagen (inhibitors of prolyl hydroxylase), Bcell antagonists (rituximab), integrin/adhesion molecule antagonists(molecules that block α1β1 and αvβ6 integrins, as well as inhibitors ofintegrin-linked kinase, and antibodies specific for ICAM-I and VCAM-I),proapoptotic drugs that target myofibroblasts, MMP inhibitors(inhibitors of MMP2, MMP9, and MMP12), and T1MP inhibitors (antibodiesspecific for TIMP-1).

The TβRII polypeptide and the co-therapeutic agent or co-therapy can beadministered in the same formulation or separately. In the case ofseparate administration, the TβRII polypeptide can be administeredbefore, after, or concurrently with the co-therapeutic or co-therapy.One agent may precede or follow administration of the other agent byintervals ranging from minutes to weeks. In embodiments where two ormore different kinds of therapeutic agents are applied separately to asubject, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that thesedifferent kinds of agents would still be able to exert an advantageouslycombined effect on the target tissues or cells.

10. Pharmaceutical Compositions

The therapeutic agents described herein (e.g., TβRII fusionpolypeptides) may be formulated into pharmaceutical compositions.Pharmaceutical compositions for use in accordance with the presentdisclosure may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Such formulationswill generally be substantially pyrogen-free, in compliance with mostregulatory requirements.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this disclosure is in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the TβRII signalingantagonists which may also optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject compounds (e.g., TβRII polypeptides) in the methodsdisclosed herein.

Typically, protein therapeutic agents disclosed herein will beadministered parentally, and particularly intravenously orsubcutaneously. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more TβRII polypeptides incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the disclosure include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions and formulations may, if desired, be presented in apack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site. In certain embodiments, compositionsof the present invention may include a matrix capable of delivering oneor more therapeutic compounds (e.g., TβRII polypeptides) to a targettissue site, providing a structure for the developing tissue andoptimally capable of being resorbed into the body. For example, thematrix may provide slow release of the TβRII polypeptides. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalcium phosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., TβRII fusionpolypeptides). The various factors include, but are not limited to, thepatient's age, sex, and diet, the severity disease, time ofadministration, and other clinical factors. Optionally, the dosage mayvary with the type of matrix used in the reconstitution and the types ofcompounds in the composition. The addition of other known growth factorsto the final composition, may also affect the dosage. Progress can bemonitored by periodic assessment of bone growth and/or repair, forexample, X-rays (including DEXA), histomorphometric determinations, andtetracycline labeling.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of TβRII fusion polypeptides. Such therapywould achieve its therapeutic effect by introduction of the TβRIIpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of TβRII polynucleotide sequences can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Preferred for therapeutic delivery of TβRIIpolynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the TβRII polynucleotide. In a preferred embodiment,the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for TβRII polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

The disclosure provides formulations that may be varied to include acidsand bases to adjust the pH; and buffering agents to keep the pH within anarrow range.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments of thepresent invention, and are not intended to limit the invention.

Example 1. Generation of Receptor Fusion Protein Variants TβRII ECDVariants

TβRII fusion proteins comprising a soluble extracellular portion ofhuman TβRII and a humanFc portion were generated. For each fusionprotein, a TβRII amino acid sequence having the amino acid sequence ofSEQ ID NO: 18 was fused to an IgG Fc portion having the amino acidsequence of SEQ ID NO: 20 by means of one of several different linkers.Each of the fusion proteins also included a TPA leader sequence havingthe amino acid sequence of SEQ ID NO: 23 (below).

Tissue plasminogen activator (TPA): (SEQ ID NO: 23)MDAMKRGLCCVLLLCGAVFVSP

An illustration summary of several of the constructs designed isprovided as FIG. 3. A table detailing the sequences for the differentconstructs tested in the Exemplification section is provided below:

Construct Amino Acid Construct Name Sequence Linker Sequence hTβRII-hFcSEQ ID NO: 9 TGGG (SEQ ID NO: 3) hTβRII (G4S)2- SEQ ID NO: 15TGGGGSGGGGS hFc (SEQ ID NO: 4) hTβRII (G4S)3- SEQ ID NO: 11TGGGGSGGGGSGGGGS hFc (SEQ ID NO: 5) hTβRII (G4S)4- SEQ ID NO: 13TGGGGSGGGGSGGGGSGGGGS hFc (SEQ ID NO: 6) hTβRII extended SEQ ID NO: 17TGGGPKSCDK hinge-hFc (SEQ ID NO: 7) hTβRII (G4S)5- SEQ ID NO: 44TGGGGSGGGGSGGGGSGGGGS hFc GGGGS (SEQ ID NO: 25) hTβRII (G4S)6-SEQ ID NO: 45 TGGGGSGGGGSGGGGSGGGGS hFc GGGGSGGGGS (SEQ ID NO: 26)

The amino acid sequences for the construct components and each of theconstructs, along with the nucleic acid sequence used to express theseconstructs, are provided below.

TβRII Portion: Amino Acid Sequence (SEQ ID NO: 18) 1TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF 51PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEYNTSN PDFc Portion: Amino Acid Sequence (SEQ ID NO: 20) 1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HNHYTQKSLS LSPGK hTβRII-hFc: Nucleic Acid Sequence(SEQ ID NO: 8) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGTGGA ACTCACACAT GCCCACCGTG CCCAGCACCT 601GAACTCCTGG GGGGACCGTC AGTCTTCCTC TTCCCCCCAA AACCCAAGGA 651CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACATGCGTG GTGGTGGACG 701TGAGCCACGA AGACCCTGAG GTCAAGTTCA ACTGGTACGT GGACGGCGTG 751GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT ACAACAGCAC 801GTACCGTGTG GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG 851GCAAGGAGTA CAAGTGCAAG GTCTCCAACA AAGCCCTCCC AGCCCCCATC 901GAGAAAACCA TCTCCAAAGC CAAAGGGCAG CCCCGAGAAC CACAGGTGTA 951CACCCTGCCC CCATCCCGGG AGGAGATGAC CAAGAACCAG GTCAGCCTGA 1001CCTGCCTGGT CAAAGGCTTC TATCCCAGCG ACATCGCCGT GGAGTGGGAG 1051AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC CCGTGCTGGA 1101CTCCGACGGC TCCTTCTTCC TCTATAGCAA GCTCACCGTG GACAAGAGCA 1151GGTGGCAGCA GGGGAACGTC TTCTCATGCT CCGTGATGCA TGAGGCTCTG 1201CACAACCACT ACACGCAGAA GAGCCTCTCC CTGTCTCCGG GTAAATGAhTβRII-hFc: Amino Acid Sequence (SEQ ID NO: 9) 1MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN 51RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG THTCPPCPAP 201ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 251EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 301EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE 351SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 401HNHYTQKSLS LSPGK hTβRII (G4S)3-hFc: Nucleic Acid Sequence(SEQ ID NO: 10) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGTGGA GGAAGTGGTG GAGGTGGTTC TGGAGGTGGT 601GGAAGTACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG 651ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 701CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC 751CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC 801CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA 851GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG 901TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 951CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT 1001CCCGGGAGGA GATGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA 1051GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC 1101GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC GACGGCTCCT 1151TCTTCCTCTA TAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG 1201AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 1251GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA ATGA hTβRII (G4S)3-hFc: Amino Acid Sequence (SEQ ID NO: 11) 1MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN 51RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201GSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 251PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 301CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK 351GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 401NVFSCSVMHE ALHNHYTQKS LSLSPGK hTβRII (G4S)4-hFc: Nucleic Acid Sequence(SEQ ID NO: 12) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGTGGA GGTTCTGGAG GTGGAGGAAG TGGTGGAGGT 601GGTTCTGGAG GTGGTGGAAG TACTCACACA TGCCCACCGT GCCCAGCACC 651TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 701ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 751GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT 801GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA 851CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT 901GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT 951CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT 1001ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG 1051ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA 1101GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG 1151ACTCCGACGG CTCCTTCTTC CTCTATAGCA AGCTCACCGT GGACAAGAGC 1201AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT 1251GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAATGAhTβRII (G4S)4-hFc: Amino Acid Sequence (SEQ ID NO: 13) 1MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN 51RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201GSGGGGSTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 251VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 301GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL 351TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 401RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence(SEQ ID NO: 50) 1 GATIPPHVQK SDVEMEAQKD EIICPSCNRT AHPLRHINND MIVTDNNGAV51 KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 101TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND 151NIIFSEEYNT SNPDTGGGGS GGGGSGGGGS GGGGSTHTCP PCPAPELLGG 201PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA 251KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 301KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP 351ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT 401 QKSLSLSPGKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence andlacking glycine prior to hTβRII portion (SEQ ID NO: 52) 1ATIPPHVQKS DVEMEAQKDE IICPSCNRTA HPLRHINNDM IVTDNNGAVK 51FPQLCKFCDV RFSTCDNQKS CMSNCSITSI CEKPQEVCVA VWRKNDENIT 101LETVCHDPKL PYHDFILEDA ASPKCIMKEK KKPGETFFMC SCSSDECNDN 151IIFSEEYNTS NPDTGGGGSG GGGSGGGGSG GGGSTHTCPP CPAPELLGGP 201SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK 251TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK 301AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 351NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ 401 KSLSLSPGKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence andlacking glycine and alanine prior to hTβRII portion (SEQ ID NO: 51) 1TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF 51PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151IFSEEYNTSN PDTGGGGSGG GGSGGGGSGG GGSTHTCPPC PAPELLGGPS 201VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT 251KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA 301KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN 351NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 401 SLSLSPGK hTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence andlacking glycine, alanine, and threonine prior to hTβRII portion(SEQ ID NO: 53) 1 IPPHVQKSDV EMEAQKDEII CPSCNRTAHP LRHINNDMIV TDNNGAVKFP51 QLCKFCDVRF STCDNQKSCM SNCSITSICE KPQEVCVAVW RKNDENITLE 101TVCHDPKLPY HDFILEDAAS PKCIMKEKKK PGETFFMCSC SSDECNDNII 151FSEEYNTSNP DTGGGGSGGG GSGGGGSGGG GSTHTCPPCP APELLGGPSV 201FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK 251PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 301GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN 351YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS 401 LSLSPGKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence andlacking glycine, alanine, threonine, and isoleucine prior to hTβRIIportion (SEQ ID NO: 54) 1PPHVQKSDVE MEAQKDEIIC PSCNRTAHPL RHINNDMIVT DNNGAVKFPQ 51LCKFCDVRFS TCDNQKSCMS NCSITSICEK PQEVCVAVWR KNDENITLET 101VCHDPKLPYH DFILEDAASP KCIMKEKKKP GETFFMCSCS SDECNDNIIF 151SEEYNTSNPD TGGGGSGGGG SGGGGSGGGG STHTCPPCPA PELLGGPSVF 201LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 251REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 301QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY 351KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 401 SLSPGK hTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence andlacking glycine, alanine, threonine, isoleucine, and proline priorto hTβRII portion (SEQ ID NO: 55) 1PHVQKSDVEM EAQKDEIICP SCNRTAHPLR HINNDMIVTD NNGAVKFPQL 51CKFCDVRFST CDNQKSCMSN CSITSICEKP QEVCVAVWRK NDENITLETV 101CHDPKLPYHD FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNIIFS 151EEYNTSNPDT GGGGSGGGGS GGGGSGGGGS THTCPPCPAP ELLGGPSVFL 201FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR 251EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 301PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK 351TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS 401 LSPGKhTβRII (G4S)4-hFc: Amino Acid Sequence lacking leader sequence andlacking glycine, alanine, threonine, isoleucine, proline, and prolineprior to hTβRII portion (SEQ ID NO: 56) 1HVQKSDVEME AQKDEIICPS CNRTAHPLRH INNDMIVTDN NGAVKFPQLC 51KFCDVRFSTC DNQKSCMSNC SITSICEKPQ EVCVAVWRKN DENITLETVC 101HDPKLPYHDF ILEDAASPKC IMKEKKKPGE TFFMCSCSSD ECNDNIIFSE 151EYNTSNPDTG GGGSGGGGSG GGGSGGGGST HTCPPCPAPE LLGGPSVFLF 201PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE 251EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP 301REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 351TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL 401 SPGKhTβRII (G4S)2-hFc: Nucleic Acid Sequence (SEQ ID NO: 14) 1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC 51AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGAGGT GGTTCTGGAG GTGGTGGAAG TACTCACACA 601TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT 651CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG 701TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC 751AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG 801GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC 851TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 901AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA 951GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA 1001CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC 1051GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA 1101GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTATAGCA 1151AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC 1201TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC 1251CCTGTCTCCG GGTAAATGA hTβRII (G4S)2-hFc: Amino Acid Sequence(SEQ ID NO: 15) 1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSTHT 201CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 251NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN 301KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS 351DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC 401SVMHEALHNH YTQKSLSLSP GKhTβRII extended hinge-hFc: Nucleic Acid Sequence (SEQ ID NO: 16) 1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC 51AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGTGGA CCCAAATCTT GTGACAAAAC TCACACATGC 601CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAG TCTTCCTCTT 651CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA 701CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC 751TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA 801GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC 851ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 901GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC 951CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA 1001AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC 1051ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC 1101CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TATAGCAAGC 1151TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC 1201GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT 1251GTCCCCGGGT AAATGA hTβRII extended hinge-hFc: Amino Acid Sequence(SEQ ID NO: 17) 1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG PKSCDKTHTC 201PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN 251WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK 301ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD 351IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS 401VMHEALHNHY TQKSLSLSPG K hTβRII (G4S)5-hFc: Amino Acid Sequence(SEQ ID NO: 44) 1 MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN51 RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201GSGGGGSGGG GSTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT 251CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH 301QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK 351NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 401TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKhTβRII (G4S)6-hFc: Amino Acid Sequence (SEQ ID NO: 45) 1MDAMKRGLCC VLLLCGAVFV SPGATIPPHV QKSDVEMEAQ KDEIICPSCN 51RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI 101TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM 151KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDTGGG GSGGGGSGGG 201GSGGGGSGGG GSGGGGSTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 251TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 301LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 351EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF 401LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKhTβRII (G4S)5-hFc: Nucleotide Sequence (SEQ ID NO: 46) 1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC 51AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGAGGA GGTTCTGGTG GTGGAGGTTC TGGAGGTGGA 601GGAAGTGGTG GAGGTGGTTC TGGAGGTGGT GGAAGTACTC ACACATGCCC 651ACCGTGCCCA GCACCTGAAC TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC 701CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA 751TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 801GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG 851AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC 901CAGGACTGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC 951CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA GGGCAGCCCC 1001GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGAGGA GATGACCAAG 1051AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 1101CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA 1151CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA TAGCAAGCTC 1201ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT 1251GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT 1301CTCCGGGTAA ATGA hTβRII (G4S)6-hFc: Nucleotide Sequence (SEQ ID NO: 47) 1ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC 51AGTCTTCGTT TCGCCCGGCG CCACGATCCC ACCGCACGTT CAGAAGTCGG 101ATGTGGAAAT GGAGGCCCAG AAAGATGAAA TCATCTGCCC CAGCTGTAAT 151AGGACTGCCC ATCCACTGAG ACATATTAAT AACGACATGA TAGTCACTGA 201CAACAACGGT GCAGTCAAGT TTCCACAACT GTGTAAATTT TGTGATGTGA 251GATTTTCCAC CTGTGACAAC CAGAAATCCT GCATGAGCAA CTGCAGCATC 301ACCTCCATCT GTGAGAAGCC ACAGGAAGTC TGTGTGGCTG TATGGAGAAA 351GAATGACGAG AACATAACAC TAGAGACAGT TTGCCATGAC CCCAAGCTCC 401CCTACCATGA CTTTATTCTG GAAGATGCTG CTTCTCCAAA GTGCATTATG 451AAGGAAAAAA AAAAGCCTGG TGAGACTTTC TTCATGTGTT CCTGTAGCTC 501TGATGAGTGC AATGACAACA TCATCTTCTC AGAAGAATAT AACACCAGCA 551ATCCTGACAC CGGTGGAGGT GGAAGTGGTG GAGGAGGTTC TGGTGGTGGA 601GGTTCTGGAG GTGGAGGAAG TGGTGGAGGT GGTTCTGGAG GTGGTGGAAG 651TACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT 701CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG 751ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA 801GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT AATGCCAAGA 851CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC 901CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA 951GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG 1001CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG 1051GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT 1101CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA 1151ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC 1201CTCTATAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT 1251CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA 1301AGAGCCTCTC CCTGTCTCCG GGTAAATGA

The various constructs were successfully expressed in CHO cells and werepurified to a high degree of purity as determined by analyticalsize-exclusion chromatography and SDS-PAGE. The hTβRII (G4S)2-hFc,hTβRII (G4S)3-hFc, hTβRII (G4S)4-hFc, hTβRII (G4S)5-hFc and hTβRII(G4S)6-hFc proteins displayed similarly strong stability as determinedby SDS-PAGE analysis when maintained in PBS for 13 days at 37° C. ThehTβRII (G4S)2-hFc, hTβRII (G4S)3-hFc, hTβRII (G4S)4-hFc proteins werealso maintained in rat, mouse or human serum and displayed similarlystrong stability.

TβRII ECD Variants

In addition to the TβRII domains included in the fusion proteinsdescribed above (e.g., SEQ ID NO: 18), the disclosure also contemplatesfusion proteins comprising alternative TβRII domains. For example, thefusion protein may comprise the wild-type hTβRII_(short)(23-159)sequence shown below (SEQ ID NO: 27) or any of the other TβRIIpolypeptides disclosed below:

(SEQ ID NO: 27) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPD(1) The hTβRII_(short)(23-159/D110K) amino acid sequence shown below(SEQ ID NO: 36), in which the substituted residue is underlined.

(SEQ ID NO: 36) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHKFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE YNTSNPD(2) The N-terminally truncated hTβRII_(short)(29-159) amino acidsequence shown below (SEQ ID NO: 28).

(SEQ ID NO: 28) 1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEYNTSNP D(3) The N-terminally truncated hTβRII_(short)(35-159) amino acidsequence shown below (SEQ ID NO: 29).

(SEQ ID NO: 29) 1 DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC51 VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF 101MCSCSSDECN DNIIFSEEYN TSNPD(4) The C-terminally truncated hTβRII_(short)(23-153) amino acidsequence shown below (SEQ ID NO: 30).

(SEQ ID NO: 30) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE Y(5) The C-terminally truncated hTβRII_(short)(23-153/N70D) amino acidsequence shown below (SEQ ID NO: 38), in which the substituted residueis underlined.

(SEQ ID NO: 38) 1 TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSDCS51 ITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI 101MKEKKKPGET FFMCSCSSDE CNDNIIFSEE Y

Applicants also envision five corresponding variants (SEQ ID NOs: 37,33, 34, 39) based on the wild-type hTβRII_(long)(23-184) sequence shownabove and below (SEQ ID NO: 49), in which the 25 amino-acid insertion isunderlined. Note that splicing results in a conservative amino acidsubstitution (Val-41e) at the flanking position C-terminal to theinsertion.

(SEQ ID NO: 49) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEYNTSN PD(1) The hTβRII_(long)(23-184/D135K) amino acid sequence shown below (SEQID NO: 37), in which the substituted residue is double underlined.

(SEQ ID NO: 37) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHKFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEYNTSN PD(2) The N-terminally truncated hTβRII_(long)(29-184) amino acid sequenceshown below (SEQ ID NO: 33).

(SEQ ID NO: 33) 1 QKSDVEMEAQ KDEIICPSCN RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF51 CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD 101PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEY 151 NTSNPD(3) The N-terminally truncated hTβRII_(long)(60-184) amino acid sequenceshown below (same as SEQ ID NO: 29).

(same as SEQ ID NO: 29) 1 DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSITSICEKPQEVC 51 VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF 101MCSCSSDECN DNIIFSEEYN TSNPD(4) The C-terminally truncated hTβRII_(long)(23-178) amino acid sequenceshown below (SEQ ID NO: 34).

(SEQ ID NO: 34) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF51 PQLCKFCDVR FSTCDNQKSC MSNCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEY(5) The C-terminally truncated hTβRII_(long)(23-178/N95D) amino acidsequence shown below (SEQ ID NO: 39), in which the substituted residueis double underlined.

(SEQ ID NO: 39) 1 TIPPHVQKSD VEMEAQKDEI ICPSCNRTAH PLRHINNDMI VTDNNGAVKF51 PQLCKFCDVR FSTCDNQKSC MSDCSITSIC EKPQEVCVAV WRKNDENITL 101ETVCHDPKLP YHDFILEDAA SPKCIMKEKK KPGETFFMCS CSSDECNDNI 151 IFSEEY

Additional TβRII ECD variants include:

(A) The N- and C-terminally truncated hTβRII_(short)(35-153) orhTβRII_(long)(60-178) amino acid sequence shown below (SEQ ID NO: 32).

(SEQ ID NO: 32) 1 DMIVTDNNGA VKFPQLCKFC DVRFSTCDNQ KSCMSNCSIT SICEKPQEVC51 VAVWRKNDEN ITLETVCHDP KLPYHDFILE DAASPKCIMK EKKKPGETFF 101MCSCSSDECN DNIIFSEEY(B) The N- and C-terminally truncated hTβRII_(short)(29-153) amino acidsequence shown below (SEQ ID NO: 31).

(SEQ ID NO: 31) 1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEY(C) The N- and C-terminally truncated hTβRII_(long)(29-178) amino acidsequence shown below (SEQ ID NO: 35).

(SEQ ID NO: 35) 1 QKSDVEMEAQ KDEIICPSCN RTAHPLRHIN NDMIVTDNNG AVKFPQLCKF51 CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD 101PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEY

Any of the above variants (SEQ ID NO: 36, 28, 29, 30, 38, 37, 33, 34,39, 32, 31, and 35) could incorporate an insertion of 36 amino acids(SEQ ID NO: 41) between the pair of glutamate residues (positions 151and 152 of SEQ ID NO: 1, or positions 176 and 177 of SEQ ID NO: 2)located near the C-terminus of the hTβRII ECD, as occurs naturally inthe hTβRII isoform C (Konrad et al., BMC Genomics 8:318, 2007).

(SEQ ID NO: 41) GRCKIRHIGS NNRLQRSTCQ NTGWESAHVM KTPGFR

As an example, the paired glutamate residues flanking the optionalinsertion site are denoted below (underlined) for thehTβRII_(short)(29-159) variant (SEQ ID NO: 28).

(SEQ ID NO: 28) 1 QKSVNNDMIV TDNNGAVKFP QLCKFCDVRF STCDNQKSCM SNCSITSICE51 KPQEVCVAVW RKNDENITLE TVCHDPKLPY HDFILEDAAS PKCIMKEKKK 101PGETFFMCSC SSDECNDNII FSEEYNTSNP D

Fc Domain Variants

While the constructs described above were generated with an Fc domainhaving the amino acid sequence of SEQ ID NO: 20, the disclosurecontemplates hTβRII-hFc fusion proteins comprising alternative Fcdomains, including a human IgG2 Fc domain (SEQ ID NO: 42, below) orfull-length human IgG1 Fc (hG1Fc) (SEQ ID NO: 43, below). Optionally, apolypeptide unrelated to an Fc domain could be attached in place of theFc domain.

(SEQ ID NO: 42) 1 VECPPCPAPP VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201CSVMHEALHN HYTQKSLSLS PGK (SEQ ID NO: 43) 1GGPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV 51DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 101NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS 151LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK 201SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Leader Sequence Variants

While the generated constructs described above included the TPA leadersequence, alternative leader sequences may be used, such as the nativeleader sequence (SEQ ID NO: 22—below) or the honey bee melittin (SEQ IDNO: 24—below) leader sequences.

Native: (SEQ ID NO: 22) MGRGLLRGLWPLHIVLWTRIASHoney bee melittin (HBML): (SEQ ID NO: 24) MKFLVNVALVFMVVYISYIYA

Example 2. Differential Ligand Inhibition by Receptor Fusion ProteinVariants in Cell-Based Assay

Affinities of TGFβ1, TGFβ2 and TGFβ3 for hTβRII (G4S)2-hFc; hTβRII(G4S)3-hFc; hTβRII (G4S)4-hFc; hTβRII-hFc; and hTβRII extended hinge-hFcproteins were evaluated in vitro with a Biacore™ instrument, and theresults are summarized in FIGS. 4A and 4B. Each of the fusion proteinswas capable of binding TGFβ1 and TGFβ3 with high affinity, but theconstructs having linker lengths longer than or equal to (G4S)4 (SEQ IDNO: 19) were surprisingly capable of binding to both TGFβ1 and TGFβ3with higher affinity than constructs having linker lengths shorter than(G4S)4 (SEQ ID NO: 19). Binding between TGFβ2 and any of the constructswas low or transient. Deglycosylation of the constructs did not changebinding.

A reporter gene assay in A549 cells was used to determine the ability ofhTβRII-hFc variants to inhibit activity of TGFβ1, TGFβ2 and TGFβ3. Thisassay is based on a human lung carcinoma cell line transfected with apGL3(CAGA)12 reporter plasmid (Dennler et al, 1998, EMBO 17: 3091-3100)as well as a Renilla reporter plasmid (pRLCMV) to control fortransfection efficiency. The CAGA motif is present in the promoters ofTGFβ-responsive genes (for example, PAI-1), so this vector is of generaluse for factors signaling through SMAD2 and SMAD3.

On the first day of the assay, A549 cells (ATCC®: CCL-18S™) weredistributed in 48-well plates. On the second day, a solution containingpGL3(CAGA)12, pRLCMV, X-tremeGENE 9 (Roche Applied Science), and OptiMEM(Invitrogen) was preincubated, then added to Eagle's minimum essentialmedium (EMEM, ATCC®) supplemented with 0.1% BSA, which was applied tothe plated cells for incubation overnight at 37° C., 5% CO₂. On thethird day, medium was removed, and cells were incubated overnight at 37°C., 5% CO₂ with a mixture of ligands and inhibitors prepared asdescribed below.

Serial dilutions of test articles were made in a 48-well plate in assaybuffer (EMEM+0.1% BSA). An equal volume of assay buffer containing thetest ligand was added to obtain a final ligand concentration equal tothe EC50 determined previously. Human TGFβ1, human TGFβ2, and humanTGFβ3 were obtained from PeproTech. Test solutions were incubated at 37°C. for 30 minutes, then a portion of the mixture was added to all wells.After incubation with test solutions overnight, cells were rinsed withphosphate-buffered saline, then lysed with passive lysis buffer (PromegaE1941) and stored overnight at −70° C. On the fourth and final day,plates were warmed to room temperature with gentle shaking. Cell lysateswere transferred in duplicate to a chemiluminescence plate (96-well) andanalyzed in a luminometer with reagents from a Dual-Luciferase ReporterAssay system (Promega E1980) to determine normalized luciferaseactivity.

As illustrated in FIGS. 5A-5F, the hTβRII (G4S)2-hFc; hTβRII (G4S)3-hFc;hTβRII (G4S)4-hFc; hTβRII (G4S)5-hFc; hTβRII (G4S)6-hFc; hTβRII-hFc; andhTβRII extended hinge-hFc proteins all were capable of inhibiting bothTGFβ1 and TGFβ3. Interestingly, while there was a correlation betweenimproved TGFβ1 and TGFβ3 inhibition and linker length for the the hTβRII(G4S)2-hFc; hTβRII (G4S)3-hFc and hTβRII (G4S)₄-hFc constructs (FIG.5E), this improvement trend appeared to have plateaued for hTβRII(G4S)5-hFc and hTβRII (G4S)6-hFc constructs (FIG. 5F).

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A Transforming Growth Factor-β Receptor II (TβRII) fusion polypeptidecomprising: a) an extracellular domain of a TβRII portion comprising anamino acid sequence that is at least 85% identical to the amino acids23-184 of SEQ ID NO: 2; b) a heterologous portion, and c) a linkerportion that comprises (GGGGS)_(n), wherein n=>4. 2-7. (canceled)
 8. Thepolypeptide of claim 1, wherein the TWIT extracellular domain portioncomprises an amino acid sequence at least 90% identical to the aminoacids 23-184 of SEQ ID NO:
 2. 9. The polypeptide of claim 1, wherein theTWIT extracellular domain portion comprises an amino acid sequence atleast 95% identical to the amino acids 23-184 of SEQ ID NO:
 2. 10. Thepolypeptide of claim 1, wherein the TβRII extracellular domain portioncomprises an amino acid sequence at least 97% identical to the aminoacids 23-184 of SEQ ID NO:
 2. 11. The polypeptide of claim 1, whereinthe TβRII extracellular domain portion comprises the amino acids 23-184of SEQ ID NO:
 2. 12. The polypeptide of claim 1, wherein the TβRIIextracellular domain portion comprises an amino acid sequence at least85% identical to SEQ ID NO:
 18. 13. The polypeptide of claim 1, whereinthe TβRII extracellular domain portion comprises an amino acid sequenceat least 90% identical to SEQ ID NO:
 18. 14. The polypeptide of claim 1,wherein the TβRII extracellular domain portion comprises an amino acidsequence at least 95% identical to SEQ ID NO:
 18. 15. The polypeptide ofclaim 1, wherein the TβRII extracellular domain portion comprises anamino acid sequence at least 97% identical to SEQ ID NO:
 18. 16. Thepolypeptide of claim 1, wherein the TβRII extracellular domain portioncomprises the amino acid sequence of SEQ ID NO:
 18. 17-31. (canceled)32. The polypeptide of claim 1, wherein the polypeptide comprises anN-terminal leader sequence.
 33. (canceled)
 34. The polypeptide of claim32, wherein the N-terminal leader sequence comprises the amino acidsequence of SEQ ID NO:
 23. 35. The polypeptide of claim 1, wherein theheterologous portion is an immunoglobulin Fc domain.
 36. The polypeptideof claim 35, wherein the immunoglobulin Fc domain is a humanimmunoglobulin Fc domain.
 37. The polypeptide of claim 35, wherein theheterologous portion comprises an amino acid sequence that is at least85% identical to SEQ ID NO:
 20. 38. The polypeptide of claim 35, whereinthe heterologous portion comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO:
 20. 39. The polypeptide of claim 35,wherein the heterologous portion comprises an amino acid sequence thatis at least 95% identical to SEQ ID NO:
 20. 40. The polypeptide of claim35, wherein the heterologous portion comprises an amino acid sequencethat is at least 97% identical to SEQ ID NO:
 20. 41. The polypeptide ofclaim 35, wherein the heterologous portion comprises the amino acidsequence of SEQ ID NO:
 20. 42-52. (canceled)
 53. The polypeptide ofclaim 1, wherein the linker comprises the amino acid sequence of SEQ IDNO:
 6. 54-71. (canceled)
 72. The polypeptide of claim 1, wherein thepolypeptide comprises an amino acid sequence that is at least 85%, 90%,95%, 97%, or 99% identical to the amino acid sequence of SEQ ID NO: 48.73. The polypeptide of claim 1, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:
 48. 74. (canceled)
 75. The polypeptideof claim 1, wherein the polypeptide includes one or more modified aminoacid residues selected from: a glycosylated amino acid, a PEGylatedamino acid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, an amino acid conjugated to a lipid moiety, andan amino acid conjugated to an organic derivatizing agent. 76-81.(canceled)
 82. The polypeptide of claim 1, wherein the polypeptideinhibits TGFβ1 with an IC₅₀ of between 0.02 nM and 1.0 nM, as determinedusing a reporter gene assay, or wherein the polypeptide inhibits TGFβ3with an IC₅₀ of between 0.02 nM and 1.0 nM, as determined using areporter gene assay. 83-89. (canceled)
 90. The polypeptide of claim 82,wherein the reporter gene assay is a CAGA reporter assay. 91-97.(canceled)
 98. A pharmaceutical preparation comprising the polypeptideof claim 1 and a pharmaceutically acceptable excipient. 99-117.(canceled)
 118. The polypeptide of claim 1, wherein the polypeptidecomprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or99% identical to the amino acid sequence of SEQ ID NO:
 52. 119. Thepolypeptide of claim 1, wherein the polypeptide comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, or 99% identical to theamino acid sequence of SEQ ID NO:
 53. 120. The polypeptide of claim 1,wherein the polypeptide comprises an amino acid sequence that is atleast 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence ofSEQ ID NO:
 54. 121. The polypeptide of claim 1, wherein the polypeptidecomprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, or99% identical to the amino acid sequence of SEQ ID NO: 55.